Degenerative Neurological and Neuromuscular Disease Dovepress

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Degenerative Neurological and Neuromuscular Disease 2012:2 141–164

Degenerative Neurological and Neuromuscular Disease

Recent advances in Duchenne muscular dystrophy

Kelly J Perkins1,2

Kay E Davies2

1Sir William Dunn School of Pathology, 2MRC Functional Genomics Unit, University of Oxford, Oxford, UK

Correspondence: Kay E Davies MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3PT, UK Tel +44 1865 285 880 Fax +44 1865 285 878 Email [email protected]

Abstract: Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-

wasting disease, is one of the most common single-gene disorders in the developed world.

Despite knowledge of the underlying genetic causation and resultant pathophysiology from lack

of dystrophin protein at the muscle sarcolemma, clinical intervention is currently restricted to

symptom management. In recent years, however, unprecedented advances in strategies devised

to correct the primary defect through gene- and cell-based therapeutics hold particular promise

for treating dystrophic muscle. Conventional gene replacement and endogenous modification

strategies have greatly benefited from continued improvements in encapsidation capacity,

transduction efficiency, and systemic delivery. In particular, RNA-based modifying approaches

such as exon skipping enable expression of a shorter but functional dystrophin protein and rapid

progress toward clinical application. Emerging combined gene- and cell-therapy strategies also

illustrate particular promise in enabling ex vivo genetic correction and autologous transplantation

to circumvent a number of immune challenges. These approaches are complemented by a vast

array of pharmacological approaches, in particular the successful identification of molecules that

enable functional replacement or ameliorate secondary DMD pathology. Animal models have

been instrumental in providing proof of principle for many of these strategies, leading to several

recent trials that have investigated their efficacy in DMD patients. Although none has reached

the point of clinical use, rapid improvements in experimental technology and design draw this

goal ever closer. Here, we review therapeutic approaches to DMD, with particular emphasis on

recent progress in strategic development, preclinical evaluation and establishment of clinical

efficacy. Further, we discuss the numerous challenges faced and synergistic approaches being

devised to combat dystrophic pathology effectively.

Keywords: dystrophy, animal models, pharmacological, exon skipping, gene therapy,

utrophin

IntroductionDuchenne muscular dystrophy (DMD) is the most common fatal genetic disorder

diagnosed in childhood, with a sex-linked inheritance pattern of one in 3500 live

male births.1,2 Affected individuals can be diagnosed at birth on the basis of elevated

serum creatine kinase (CK), a biochemical marker of muscle necrosis,3 prior to visible

difficulty in walking between 1 and 3 years of age. The clinical course of DMD is

progressive; muscle weakness by age 5 years eventually leads to loss of independent

ambulation by the middle of the second decade and death during the third decade,

primarily as a result of respiratory or cardiac complications.2 The genetic causation of

DMD was established by localization of candidate complementary DNAs (cDNAs) to

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Degenerative Neurological and Neuromuscular Disease 2012:2

the short arm of the X chromosome (Xp band 21.2),4 which

led to full characterization of the 2.5-Mb DMD locus and

corresponding 427-kDa dystrophin protein.5 The sheer size

of the resulting 14-kb dystrophin messenger RNA transcript

served to explain how one-third of DMD cases arise from

spontaneous new mutations.6 In terms of clinical manifesta-

tion, DMD results from failure to produce functional dys-

trophin protein as a result of nonsense or frame-shift DNA

mutations,6 whereas those retaining the amino acid reading

frame result in partially functional dystrophin and the milder

allelic variant, Becker muscular dystrophy (BMD).

The genetic link to dystrophic pathology was elucidated

by localization of dystrophin protein to the sarcolemma

of skeletal and cardiac muscle, which is absent in DMD

patients.5 Structural and functional studies illustrate that

dystrophin is pivotal for maintaining structural integrity by

linking the internal actin cytoskeleton of individual muscle

fibers via F-actin binding of its N-terminus7 and C-terminal

binding to the dystrophin-associated protein complex

(DAPC) through β-dystroglycan (β-DG).8 The DAPC com-

prises several internal scaffold and transmembrane proteins,

including α/β-DGs, sarcoglycans, sarcospan, and biglycan,

by which linkage to collagen and laminin is achieved; while

evidence suggests some of these links have functional sig-

naling roles, their predominant purpose appears mechanical

(reviewed in Davies and Nowak).9 In addition, the C-terminus

of dystrophin interacts with neuronal nitric oxide synthase,10

dystrobrevin,11 and the syntrophins.12 At the molecular level,

loss of dystrophin and consequential loss of the DAPC create

sarcolemmal instability, enhancing susceptibility to mechani-

cally induced damage and degeneration.13 Although the

muscle initially responds through enhanced regeneration,14

successive rounds of necrosis eventually deplete the sup-

ply of muscle progenitor cells, which leads to infiltration

of adipose and fibrotic connective tissue and exacerbates

muscle wasting.15

Fragility of the DAPC also results in stretch-induced

membrane permeability, leading to disruption of cellular

homeostasis.16 The resultant elevation of intracellular

calcium ([Ca2+]i) levels triggers increased production of

reactive oxygen species (ROS) by the mitochondria,17 which

contributes to the self-perpetuating cycle of increased oxi-

dative stress, sarcolemmal damage, and eventual myofiber

death.6 Chronic activation of signaling pathways involved

in the inflammatory response further exacerbates dystro-

phic pathology by increasing myofiber expression of the

major histocompatibility complex18 and the secretion of

chemokines and cytokines.19 Although profibrotic signaling

is initially activated in an attempt to repair compromised

myofibers, it is susceptible to upregulation by the unremit-

ting nature of muscle damage, which triggers fibrosis and

perpetuates inflammation.20 As the cellular mechanisms that

govern these secondary responses are intimately linked, it is

difficult to ascribe hierarchical order to the molecular events

that exacerbate DMD pathogenesis.

Although standards of care are improving, with better

quality of life and prolonged survival,3 there is no cure

for DMD. Clinical intervention is generally restricted to

symptom management, such as ventilators for respiratory

support and administration of glucocorticoids to stem pro-

gressive muscle damage. Long-term corticosteroid treatment

purportedly extends functional ability for up to 2 years21 by

modifying cellular events, including inflammation and Ca2+

homeostasis; however, their relative nonspecificity also

causes unfavorable effects such as weight gain and loss of

bone density.22 Nonetheless, established steroidal efficacy

provides a basis for devising therapeutic strategies able

specifically to target molecular defects underlying dystrophic

pathology. Several promising approaches have emerged due

to advances in experimental design, delivery, and efficacy

for all three subgroups: gene therapy, cell therapy, and phar-

macological therapy. In this review, we describe the current

status of each approach, with particular emphasis on clinical

application. Further, we discuss emerging combinatorial

strategies that are most likely to provide future candidates

for a definitive DMD therapy.

Mammalian models of Duchenne muscular dystrophyAnimal models have been an invaluable resource to elucidate

the molecular basis of DMD pathogenesis and in assessing

therapies that may carry substantial risk in humans (Table 1).

As the dystrophin-deficient phenotype significantly differs

between species, the suitability of each animal model is pri-

marily based on phenotypical similarity to DMD, weighed

against the extent of pathological characterization, scope for

genetic manipulation, accessibility, and breeding costs.23

Murine models of DMDMouse models are indispensable for developing therapeutic

approaches for DMD, since they are easily and reliably

reproduced. The widely used X-linked muscular dystrophy

mouse mdx model24 arises from a spontaneous nonsense

mutation in exon 2325 and absence of dystrophin protein.5

Although muscle necrosis and high CK levels are evident

from 2 weeks, the mdx phenotype is most pronounced


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between 3 and 4 weeks, when, in contrast to DMD patients,

successive cycles of extensive necrosis are countered by

regeneration,24 eventually decreasing to chronic low-level

damage by 8 weeks, permitting a near-normal lifespan.23

Further, deterioration of skeletal and cardiac muscle (includ-

ing fibrosis and inflammatory cell infiltration at later stages)

in mdx is comparatively mild, where only the diaphragm is

considered to recapitulate the severity of human disease.26

This phenotypic disparity extends to N-ethyl-N-nitrosourea

(ENU)-induced genetic variant mdx strains (mdx2–5cv)27

that are not commonly used for therapeutic studies. Despite

issues involving body size, genetic background, and patho-

logical features, mdx is the established model for in vivo

efficacy due to its desired transgenic and breeding capacity.

For example, gene-based skipping of exon 51 (a strategy

that is theoretically applicable to the highest percentage of

DMD patients with out-of frame deletion mutations)28 can

be assessed using exon 52 knockout mice (mdx52).29 Further,

the development of “humanized” (hDMD) transgenic mice

containing full-length human dystrophin has recently enabled

direct preclinical screening of human-specific exon-skipping

approaches.30

Although inaccurate as genetic models, several double

knockouts, including the myogenic transcription factor

MyoD,31 the discriminant analysis of principal component

(DAPC) α-DB,32 parvalbumin,33 α7 integrin,34 cytidine

monophosphate–sialic acid hydroxylase,35 and the dystrophin

autosomal paralogue utrophin36 have been developed. The

most clinically relevant and widely used are dystrophin/

utrophin knockout mice (mdx; utrn–/–, commonly referred

to as dko),36 which illustrate similar pathology to mdx at

4–5 weeks, after which this model progressively recapitulates

DMD disease pathogenesis, resulting in a dramatically

reduced lifespan.23 As decreased survival of dko mice poten-

tially hampers experimental design, a haploinsufficiency

model (mdx; utrn+/–) has been generated but is not widely

used.37

Canine X-linked muscular dystrophySpontaneous canine X-linked muscular dystrophy (CXMD)

has been reported38 in golden retriever (GRMD),39 beagle

(CXMDJ),40 rottweiler,41 German shorthaired pointer,42

Japanese spitz,43 and Cavalier King Charles spaniel

( CKCS-MD)44 breeds. Of these, the phenotypic progres-

sion of GRMD, resulting from an intron 6 splice acceptor

mutation (leading to skipping of exon 7 and truncated dys-

trophin protein)39 has been the most extensively character-

ized.23 GRMD represents the most accurate animal model

for DMD in recapitulating phenotypic timing and severity,

where muscle degeneration and generalized necrosis noted

from birth onwards results in extensive fibrosis by 6 months

and respiratory failure commensurate to human pediatric

age.39 Given the retriever’s suitability in respect of genetic

background and body size, GRMD has been instrumental

in predicting disease pathogenesis, severity, and treat-

ment efficacy,45 providing proof of concept for numerous

cell- and gene-therapy approaches (see Table 1). However,

the use of GRMD is restricted by dramatic phenotypical

variation between sibs (causing difficulties in preclinical

standardization),39 welfare implications, and high costs of

maintenance and treatment.23 These concerns have been par-

tially addressed by interbreeding GRMD dogs with smaller

beagle sires (canine X-linked muscular dystrophy in Japan

[CXMDJ]), resulting in a near-identical phenotype to GRMD

but with an improved survival rate.46

Although GRMD and CXMDJ47 dogs have several advan-

tages over mdx as an exon-skipping model, they also retain

a similar disadvantage where the disease-causing mutation

lies outside the region commonly affected in humans.48 The

recent characterization of severe CXMD in CKCS dogs is

of particular clinical relevance given its genotypic causation

(a donor splice acceptor mutation in exon 50 and predicted

protein truncation).44 Further, success in inducing exon

51 skipping in cultured CKCS-MD myoblasts44 indicates

the potential of CKCS-MD as a suitable in vivo model (see

gene-therapy section).

Feline and porcine modelsHypertrophic feline muscular dystrophy (HFMD)49 and the

238 tailored pig model (238-DMD)50 represent two large

animal models of DMD that substantially differ in their

genetic derivation that are suitable candidates for therapeutic

assessment. While HFMD represents spontaneous dystrophin

deficiency as a result of an extensive promoter deletion,51

it is not widely used to limited pathological similarity to

DMD.38 In contrast, the exon 52–deleted 238-DMD pig,

similar to mdx52, was engineered to assess exon 51 skip-

ping methodologies,50 and appears to be a bona fide model,

as ascertained by absence of dystrophin protein, elevated

serum CK levels, and early degenerative changes on muscle

histology.50 Further, porcine models have a number of practi-

cal advantages, such as the ability to circumvent numerous

issues that currently preclude experimental transition from

mdx into larger models (such as transgenic manipulation and

breeding considerations), while retaining a similar size and

physiology to humans.


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DMD: an overview of therapeutic approaches

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Tab

le 1

Ani

mal

mod

els

used

in a

sses

sing

the

rape

utic

str

ateg

ies

for

Duc

henn

e m

uscu

lar

dyst

roph

y (D

MD

)

mod

elP

rovi

denc

eM

utat

ion

Pat

holo

gy a

nd

com

men

tsG

ene

and

prot

ein

repl

acem

ent

Gen

e re

pair

and

ex

on s

kipp

ing

Cel

l/pha

rmac

olog

ical

the

rapy

mdx

24§

mou

seSp

onta

neou

s31

85C

.T

(e

xon

23)

nons

ense

25

Mus

cle

fiber

de

gene

ratio

n/

rege

nera

tion,

m

yopa

thy

less

se

vere

late

r in

lif

e, n

orm

al

lifes

pan24

us

ed fo

r m

ost

stra

tegi

es o

win

g to

bre

edin

g an

d ex

peri

men

tal

sim

plic

ity23

‡

Gen

e re

plac

emen

t**

aden

ovir

us: m

ini-

(mD

YS)

, mic

ro-(

mDY

S)

dyst

roph

in.77

‡ C

ell/g

ene

repl

acem

ent

lent

ivir

al: m

DY

S-M

DSC

91,

plas

mid

: mD

YS-

MD

SC95

-

myo

blas

t96,9

7 DY

S-

HA

C(F

L)-iP

S100 ,

MA

B101

Pro

tein

com

pens

atio

n ut

roph

in (

TA

T-m

Utr

)180 ,

bigl

ycan

(rh

BGN

)185 ,

lam

inin

(LA

M-1

11)19

3,19

4 ut

roph

in u

preg

ulat

ion

Gen

e re

pair

102,

103

exon

ski

ppin

g 2’

OM

ePS/

PMO

† , 2’

OM

ePS/

PMO

M

STN

273 2

’OM

ePS

MST

N/D

YS27

4

Cel

l: al

loge

nic

hum

an a

nd m

dx

myo

blas

ts‡5

5 allo

geni

c m

urin

e an

d hu

man

MD

SC62

-64*

Pha

rmac

olog

ical

: D

ystr

ophi

n ge

ne: n

egam

ycin

, ge

ntam

icin

147,

148,

152 f

unct

iona

l co

mpe

nsat

ion/

mem

bran

e st

abili

ty:

utro

phin

† , α7

inte

grin

191,

192 ,

PA18

8200 ,

RO

S in

hibi

tors

: an

tioxi

dant

s† , BG

P-15

215 ,

Nec

rosi

s:

PDE5

inhi

bito

rs23

0,23

1 , N

O-

rele

asin

g ag

ents

† MPT

P/ca

lpai

n in

hibi

tors

†

Fibr

osis

: TG

F-β/

BMP/

RA

S an

tago

nist

s† MST

N in

hibi

tion‡2

55

infla

mm

atio

n: p

redn

isol

one† ,

TN

F-α/

NF-

κB in

hibi

tion16

2‡, i

GF-

1†

mdx

4Cv27

m

ouse

ENU

m

utag

enes

is79

25C

.T

(e

xon

53)

nons

ense

10 t

imes

few

er

fiber

rev

erta

nts

than

mdx

-2C

v w

ith e

ssen

tially

id

entic

al m

uscl

e pa

thol

ogy27

Gen

e re

plac

emen

t pl

asm

id: m

DY

S86,

aden

ovir

us: m

DY

S77‡

Cel

l/gen

e re

plac

emen

t le

ntiv

irus

: mD

YS-

SC92

Exo

n sk

ippi

ng

2’O

MeP

S/PM

O11

7 M

ultip

le

Non

e re

port

ed

mdx

5Cv27

m

ouse

ENU

m

utag

enes

is13

06a.

t SD

(e

xon

10)

mis

sens

e

Path

olog

y as

per

m

dx4C

v27

Cel

l/gen

e re

plac

emen

t le

ntiv

irus

: mD

YS-

MD

SC90

Gen

e re

pair

107,

108

Pha

rmac

olog

ical

RO

S in

hibi

tors

/ant

ioxi

dant

s: m

elat

onin

206

mdx

5229

m

ouse

Tar

gete

d di

srup

tion

Δexo

n 52

Sim

ilar

path

olog

y to

mdx

, with

lim

b m

uscl

e hy

pert

roph

y29

Non

e re

port

edE

xon-

skip

ping

PM

O11

5

Non

e re

port

ed

mdx

;utr

n-/-

(d

ko)36

m

ouse

Tar

gete

d di

srup

tion

mdx

:utrn

tm

1Jrs

(e

xon

7)

Non

sens

e

Con

side

red

a D

MD

phe

noco

py

over

mdx

due

to

earl

ier

onse

t of

m

uscl

e dy

stro

phy

and

prem

atur

e de

ath36

Gen

e re

plac

emen

t**

aden

ovir

us: m

ini-(

mD

YS)

an

d m

icro

-(mD

YS)

dy

stro

phin

77‡

Pro

tein

com

pens

atio

n ut

roph

in (

TA

T-m

Utr

)180 ,

α7 in

tegr

in p

eptid

e189

Exo

n-sk

ippi

ng

in v

ivo:

PM

O

aden

ovir

us

U7s

nRN

A13

1

Cel

l Le

ntiv

iral

sys

tem

ic a

lloge

nic

(don

or d

eriv

ed)

and

auto

logo

us

MA

B73

Pha

rmac

olog

ical

M

embr

ane

stab

ility

: vPA

191 ,

ROS

inhi

bito

rs: B

GP-

1521

5 , fib

rosi

s:

RA

S†267

, BM

P263 ,

TG

F-β24

9 an

tago

nist

s, in

flam

mat

ion:

IGF-

1277


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hDM

D30

m

ouse

YA

C in

sert

ion

of fu

ll-le

ngth

hu

man

dy

stro

phin

30

Tel

omet

ric

(chr

5)

inte

grat

ion13

9

Com

plem

ents

dy

stro

phic

pa

thol

ogy

in m

dx

(hD

MD

/ mdx

)30

and

dko

cros

ses

(hD

MD

/dko

)139

Not

app

licab

leE

xon-

skip

ping

in

viv

o: 2

’OM

ePS

sing

le11

5 mul

tiple

30,

2’O

MeP

S / P

MO

si

ngle

124 ,

aden

ovir

us -

U

7snR

NA

mul

tiple

132

Not

app

licab

le

GR

MD

39

gold

en

Ret

riev

er

Spon

tane

ous

739-

2a.

g39

SA (

intr

on 6

) de

letio

n

Mos

t si

mila

r to

D

MD

pat

holo

gy

of a

ll m

odel

s23

limita

tion

of

phen

otyp

ic

vari

atio

n be

twee

n si

bs39

Gen

e re

plac

emen

t ad

enov

irus

: min

i-(m

DY

S)

dyst

roph

in77

,81‡

, min

iutr

ophi

n (m

Utr

)77‡8

0 C

ell/g

ene

repl

acem

ent

lent

ivir

al: m

DY

SMD

SC91

, M

AB93

Gen

e re

pair

104

exon

-ski

ppin

g in

vitr

o: 2

’OM

ePs/

PM

O s

ingl

e113‡

in

viv

o:

AA

vU

7snR

NA

si

ngle

133,

134

Cel

l Sy

stem

ic a

lloge

nic

(don

or-d

eriv

ed)

cani

ne M

AB93

P

harm

acol

ogic

al

Mem

bran

e st

abili

ty: P

A18

8201

calp

ain

inhi

bito

rs: C

10123

7

CX

MD

J40

beag

leSp

onta

neou

s73

9-2a

.g39

SA

(in

tron

6)

dele

tion

Phen

otyp

e as

per

G

RM

D; s

mal

ler

bree

d si

ze40

Gen

e re

plac

emen

t ad

enov

irus

: min

i-(m

DY

S)

dyst

roph

in82

Exo

n-sk

ippi

ng

in v

itro:

PM

O

sing

le11

3 in

vivo

: 2’

OM

ePS/

PMO

M

ultip

le47

,116

Cel

l Sy

stem

ic/in

tram

uscu

lar

allo

geni

c (d

onor

-der

ived

)72

CK

CSM

D44

sp

anie

lSp

onta

neou

s72

94+5

t.g

SD (

intr

on 5

0)

dele

tion

Phen

otyp

e as

per

G

RM

D; s

mal

ler

bree

d si

ze44

Non

e re

port

edE

xon-

skip

ping

ce

ll ba

sed:

PM

O

sing

le44

Non

e re

port

ed

Not

es: ‡ O

verv

iew

of e

xper

imen

tal a

ppro

ach/

path

olog

y; † m

ultip

le s

tudi

es: r

efer

to r

elev

ant s

ectio

ns; *

usin

g m

dx/s

ever

e co

mbi

ned

imm

unod

efici

ency

mic

e; *

*dys

trop

hin

cons

truc

ts o

nly;

# hum

an s

peci

fic s

eque

nce.

App

roac

hes

not r

epor

ted

for

§ mou

se: m

dx v

aria

nts:

mdx

-2cv

27 (6

326+

2a.

t)-3

cv27

(977

2–16

t.a)

; ‡ can

ine:

(se

e38),

rott

wei

ler41

, Ger

man

poi

nter

42 a

nd Ja

pane

se s

pitz

43, o

ther

: fel

ine

HFM

D m

odel

(de

l Dp4

27m

+Dp4

27p49

) an

d pi

g D

MD

-238

(Δe

xon

52)50

.A

bbre

viat

ions

: SA

/SD

, spl

ice

acce

ptor

/spl

ice

dono

r; E

NU

, N-e

thyl

-N-n

itros

oure

a; S

C, s

atel

lite

cell;

2′O

MeP

S, 2

′-O-m

ethy

l olig

orib

onuc

leot

ide;

PM

O, p

hosp

horo

diam

idat

e m

orph

olin

o ol

igom

er; i

PS, i

nduc

ed p

luri

pote

nt s

tem

cel

ls; F

L,

full

leng

th; r

hBG

N, r

ecom

bina

nt h

uman

big

lyca

n; M

STN

, myo

stat

in; M

DSC

, mus

cle

deri

ved

stem

cel

ls; M

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acid

.


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As there is no definitive DMD animal model, GRMD

and mdx currently represent the most appropriate model for

preclinical testing by consensus.23 It is tempting to speculate

reliance on the mdx mouse model has hampered therapeutic

progress, as the mild clinical phenotype results in difficulty in

assessing certain issues, such as devising gene or cell therapies

for larger muscle.52 Nonetheless, the genetic tractability, repro-

ducibility, and convenient size make mouse models invaluable

tools in DMD research, provided their physiological differ-

ences are acknowledged. This reliance on smaller animals is

largely due to practical difficulties imposed by larger models

such as GRMD, which more closely represent DMD patients

in size and pathological expression, but are never likely to

supersede mdx in high-throughput studies. However, the future

use of dog (and possibly pig) models to hone mouse-developed

technologies on a more comparable phenotype is unclear,

given a number of proof-of-concept strategies developed

in mdx and human DMD cell lines have circumvented their

use to successfully progress to human safety trials.

Cell-based therapeutic approachesCell-based therapies involve transplantation or transduction

of allogeneic (donor-derived) or autologous (patient-derived)

cells to engraft with existing myofibers or repopulate the cel-

lular niche to promote functional muscle regeneration.53

Myoblast transplantationAllogeneic myoblast transfer was the f irst cell-based

strategy to be assessed in immunologically tolerant mice,

providing evidence of host–donor fusion and stimulating

myofiber development;54 parameters that were subsequently

recapitulated in mdx mice (reviewed in Mouly et al).55

Although allogeneic cell transplantation can circumvent

the need for genetic manipulation to reintroduce functional

dystrophin, the risk of graft rejection remains.57 In addition,

several unfavorable characteristics of using donor myoblasts,

including (1) poor intramuscular migration, (2) low efficiency

of dystrophin production, (3) limited cell survival, and

(4) immune complications in mdx, were mirrored in early

clinical trials assessing allogeneic implantation of immuno-

histocompatible myoblasts in DMD patient muscle.56 Further,

this approach leads to massive central ischemic necrosis in

nonhuman primates.57

Satellite cells and muscle-derived stem cellsAs a result of the various pitfalls encountered with myoblast

transfer, stem cell transplantation was deemed a more

attractive option due to their differentiation potential and

self-renewal capacity.58 Among the first to be assessed were

satellite cells (SCs), a quiescent and committed population

of myogenic precursors that actively divide and differentiate

in response to myofiber growth or damage.59 When SCs

remain attached to single myofibers for transplantation, they

illustrate self-renewal and self-sufficiency as a regenerative

source.60 At present, direct SC engraftment faces two

major hurdles: (1) the rapid decline of their autologous

isolation potential, especially in the later stages of muscle

degeneration, and (2) their individual isolation, in particular

as in vitro expansion drastically reduces their engraftment and

regeneration capacity.61 It also remains unclear whether SCs

derive from precursors resident in muscle or from circulating

progenitors.60 A number of these parameters can be alleviated

through the use of muscle-derived stem cells (MDSCs),

which are commonly thought to represent a predecessor of the

SC.59 As MDSCs represent a multipotential cell population,

they are considered distinct from the myogenically committed

SCs. Further, MDSCs have a number of advantages over SCs,

including (1) increased engraftment ability, (2) expression

of specific stem cell markers that allow specific isolation,

and (3) expansion and maintenance in an in vitro progenitor

state.20,59,62 Systemic delivery of allogeneic murine or

human MDSCs63,64 can restore dystrophin expression and

ameliorate dystrophic pathology in immunotolerant mdx/

severe combined immunodeficiency (SCID) mice. Further,

autologous transplantation of MDSCs in DMD patients

during a Phase I clinical safety study did not result in local

or systemic side effects.62 Despite these encouraging results,

the typically heterogeneous nature of MDSCs may affect

their efficacy, depending on their isolation and culturing

conditions.59

Pluripotent and non-muscle-derived progenitor cellsSeveral non-muscle cell types such as embryonic stem (ES)

cells can converted to myogenic precursors after coculturing

with skeletal myoblasts or by myogenic induction.65 To cir-

cumvent ethical and legal restrictions associated with deriv-

ing ES cells,66 allogeneic pluripotent human cells have been

successfully isolated from early-age amniotic fluid (human

AF-amniotic fluid stem cells) and umbilical cord (human

umbilical cord-derived mesenchymal stem cells [hUC-

MSCs]). Both of these donor cell populations were able to

fuse with host myofibers after intramuscular or intravascular

delivery, respectively, in immunosuppressed mice, although

not within a dystrophic (ie, mdx) genetic background.67,68


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However, given that hUC-MSC engraftment demonstrates

effective elevation of muscle proteins in dysferlin-deficient

dystrophic mice (an animal model for limb-girdle muscular

dystrophy type 2B and Miyoshi myopathy, both caused by

mutations of the dysferlin gene),68 a planned Phase I/II trial

is currently recruiting to assess their safety and efficacy in

DMD patients.69

In recent years, a number of tissue-specific adult stem

cells, which maintain, generate, and replace terminally

differentiated cells within their resident organ, have dem-

onstrated myogenic potential.53 Among the most promising

are adult MSCs, which can differentiate to form myogenic

cells in situ.20 In contrast to other DMD cell-based therapies,

MSCs also possess distinct anti-inflammatory activities and

represent an ethical alternative to ES cells.70 For example,

intramuscular injection of bone marrow-derived MSCs

was successful in regenerating muscle cells and repairing

muscle degeneration in mdx/SCID mice.71 Intramuscular

or interarterial injection of myogenically induced canine

wild-type allogeneic (dog leukocyte antigen matched with

an unaffected littermate donor) bone marrow MSCs was

able to establish long-term, widespread muscle engraft-

ment and differentiation in CXMDJ dogs without requiring

immunosuppression.72

Another promising MSC-based approach is the use

of vessel-associated mesoangioblasts (MABs), multipo-

tential progenitors with the ability to differentiate into

many mesodermal phenotypes, including myotubes.70

Interarterial delivery of donor wild-type MABs in GRMD

dogs illustrates impressive engraftment capability, leading

to extensive recovery of muscle morphology and function.70

Encouragingly, similar parameters can be achieved in

dko mice, where proliferating MABs illustrated the ability

to form new myofibers and promote expression of dystro-

phin and its autosomal paralogue – utrophin.73 These results

establish MABs as a feasible candidate for DMD stem cell

therapy, and an interarterial Phase I/IIa DMD clinical trial

using MABs from healthy donors has been initiated.74

From myoblast transfer to the use of stem and progenitor

cells, a common hurdle remains in the effective use of cell

therapy in DMD patients. For cell transplantation to be suc-

cessful, hurdles such as immune rejection must be overcome,

or advances in developing methods to manipulate autologous

cells to reexpress dystrophin must be made. The most prom-

ising cell-based approach is thus likely to involve ex-vivo

expansion of pluripotent patient-derived myogenic precursors

(such as MABs) with gene therapy to enable autologous,

genetically corrected cell engraftment. Recent progress in

the rapidly expanding combined cell–gene therapy field is

outlined in the next section.

Gene-based therapeutic approachesAs DMD is caused by recessive and monogenic genetic

mutations, therapeutic strategies can be devised to correct

or improve muscle function by (1) exogenous delivery

of functionally engineered dystrophin gene constructs or

(2) repair/augmentation of the endogenous locus (Figure 1).

Encouragingly, both approaches benefit from either the rapid

progress in RNA-based technology or by combination with

cell-based therapies.

Gene-replacement therapyDelivery of exogenous functional dystrophin is an attractive

prospect to benefit all DMD patients (given the inconsequen-

tial nature of the endogenous mutation), and gene replace-

ment is traditionally divided into viral and naked (nonviral)

categories. The major challenge common to viral and nonviral

approaches involves developing suitable delivery vectors and

gene cassettes while avoiding a destructive immune response.

Further, the large size of the dystrophin gene, coupled with

the limited carrying capacity of vectors such as recombinant

adeno-associated virus (rAAV) prompted construction of

internally deleted but highly functional “mini”-dystrophin

(mDYS)75 and “micro”-dystrophin (mDYS)76 constructs to

facilitate gene transfer.

Historically, studies using systemic and intramuscular

rAAV-mediated delivery of mDYS and mDYS in mdx dem-

onstrated promising efficacy in a number of parameters,

including successful formation of sarcolemmal mDYS-/

mDYS-associated protein complexes and improved muscle

function while reducing fibrosis (reviewed in Bowles et al).77

Although in vivo rAAV-mediated gene transfer has been

effective in reducing dystrophic pathology in both GRMD

and dko animal models (reviewed in Seto et al),78 the immune

reaction against rAAV particles and dystrophin protein itself

has been readily apparent in mdx and is particularly severe in

GRMD.79 Recent efforts to improve immune tolerance and

transduction efficiency have led to increasing use of rAAV8-

and AAV9-modified serotypes. For example, intramuscular

rAAV2/9-mDYS80 and rAAV9-mDYS81 gene transfer in mdx

and systemic injection of rAAV8-mDYS in CXMDJ82 and

rAAV8-mDYS in GRMD81 not only illustrate widespread

transgene expression but also increase tropism in cardiac

and skeletal muscle. However, lingering immune concerns

continue to limit clinical assessment of rAAV-mediated gene

transfer. This was evident in a 2010 Phase I dose-escalation


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A.


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study using intramuscular rAAV2.5-mDYS injection,

which elicited failure in long-term transgene expression and

severe T-cell reaction in a small cohort of DMD patients.83

However, it is likely that AAV-mediated gene replacement

will be subject to future trials, given recent improvements

in translation optimization of the rAAV2.5 capsid has led to

vastly improved immune tolerance in a Phase I follow-up

safety study.77

Nonviral gene-transfer methodologies have been

explored by intramuscular injection of naked full-length

and mDYS plasmid into mdx hindlimb muscle,84,85 mDYS

in mdx4cv diaphragm,86 and more recently by electrotransfer

of full-length canine dystrophin into GRMD hind limb.87

These studies indicate that plasmid-based gene transfer

has greater potential for long-term expression compared

to rAAV-mediated approaches but is hampered by lower

comparative efficacy. Due perhaps to the latter, an initial

clinical report detailing success of intramuscular delivery of

full-length/mDYS plasmids in DMD patients to express func-

tional protein within the injection site88 has not been repeated.

Further, commercial development of a plasmid-based therapy

(Myodys; Transgene) was assessed in a Phase I trial in 2008;

however, no data has subsequently been released.89

An increasingly promising alternative strategy to deliver

functional dystrophin involves the ex vivo combination of

cell and gene therapies. This approach involves the use of

genetically modified cells as autologous delivery vehicles

to circumvent immune challenges and reduce the risk of

implant rejection.90 Systemic efficacy of combined cell–gene

therapy was originally established by successful interarte-

rial delivery of MDSC cells transduced with lentivirus to

regenerating mdx5cv muscle.90 This study was extended by

lentiviral-mediated transduction of canine mDYS in human

and GRMD MDSCs prior to transplantation into mdx and

GRMD by either intramuscular injection or electrotransfer.91

Lentiviral vectors have also been used to demonstrate that

mDYS-transduced autologous mdx4cv SC92 and GRMD

MABs93 can regenerate dystrophin-positive myofibers in vivo.

However, it is important to note that although the level of

mDYS-expressing fibers was sufficient in treated GRMD

dogs to ameliorate dystrophic morphology (5%–50%), their

clinical performance remained poor, in direct contrast to the

phenotypical improvement observed using systemic delivery

of unmodified donor cell MABs93 (see section on cell-based

therapeutics.).

Alternative viral delivery vehicles such as retrovirus

also demonstrate transduction efficacy, although the risk of

immunogenic graft rejection is increased.94,96 Nonetheless,

interarterial administration of isogenic MSCs containing

retroviral-induced mDYS enabled persistent, long-term

(12-week) dystrophin restoration in mdx muscle fibers and

satellite cells.94 Although plasmid-based mDYS transduction

in MDSC95 and mdx/DMD myoblasts96,97 induces high in situ

expression, mini-gene approaches have been superseded

by development of a human artificial chromosome (HAC)

containing the entire dystrophin gene (DYS-HAC).98 DYS-

HAC has a number of distinct advantages over plasmid-

based approaches, such as stable episomal maintenance and

ability to carry large genomic regions (including regulatory

elements). DYS-HAC transduction via microcell-mediated

chromosome transfer enables complete genetic correction of

engraftable induced pluripotent stem (iPS) cells99 from mdx

and DMD patients.100 Further, correct tissue expression of

human dystrophin isoforms was evidenced in chimeric mice

generated from DYS-HAC ES cell transfer.100 Efficacy has

recently been established in vivo using systemic delivery of

DYS-HAC–transduced MABs, which illustrated morphologi-

cal and functional amelioration of dystrophic pathology for

up to 8 months posttransplantation.101 These approaches have

led to planned trials of DYS-HAC–transduced MDSCs for

autologous transplantation in patients.74

Gene editingAn alternative approach to exogenous delivery of functional

dystrophin by gene replacement or cell-based therapies is to

induce de novo dystrophin production. Gene editing aims to

repair or modify the underlying genetic defect (gene repair)

or to modulate RNA processing (by selectively “skipping”

exons of the dystrophin gene) to ameliorate effects of the

underlying gene mutation.

Gene repairInitial approaches to gene editing were aimed at correcting

point mutations in the dystrophin gene using synthetic RNA/

DNA “chimeraplasts” (RDOs), which enter the cell and attach

to the target gene. The DNA strands of the chimeraplast and

the gene complement each other with the exception of the

nucleotides that require editing, which are then targeted by

DNA repair enzymes, allowing the permanent replacement

of the DNA target sequence with that of the chimeraplast.102

Although direct injection of RDOs into mdx muscle resulted in

sarcolemmal localization of full-length dystrophin, myofiber

conversion rates were poor (ranging from 1% to 15%).102,103

Similarly, direct intramuscular injection of RDOs illustrated

sustained (over 48 weeks) in vivo repair of the GRMD point

mutation and production of full-length dystrophin; resulting


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levels of dystrophin protein were similarly low, and almost

exclusively restricted to the injection site.104

Consequently, RDO-mediated editing has been superseded

by antisense oligodeoxynucleotide (AON) approaches due

to considerations such as modification ability, cost, scale,

and experimental variability.105 Although RDO-induced

point mutations can successfully enable mdx exon-skipping

in vitro,106 it is notable that AON-mediated gene editing

in vitro and in vivo using the mdx5cv mouse model107,108 was

originally devised to prevent not encourage exon skipping.

The emergence of the latter as one of the most promising

therapeutic approaches for DMD has led to a decline in using

traditional gene-editing methodologies.

Exon skippingExon skipping is the most frequent alternative splicing

mechanism known in mammals, and as such is a major

contributor to protein diversity.109 AONs aim to mimic

exclusion (or “skipping”) of specific exons by hybridizing

and thus blocking targeted pre-mRNA motifs involved in

normal splicing to synthesize an internally truncated, semi-

functional dystrophin protein.48 Exon skipping has immense

clinical potential, as 60% of DMD patients harbor deletions

in exons 45–55 and sole targeting of exon 51 can address

the majority of patients by addressing deletions of exon

50, exon 52, exons 48–50, or exons 49 and 50.48 Further, a

small-molecule “cocktail” approach enabling multiple exon

skipping can feasibly be marketed as a single drug (reviewed

in Benchaouir and Goyenvalle).110

Preclinical in vitro proof of concept for AON-mediated exon

skipping was established in primary cultured mdx myoblasts

using targeted 2′-O-methyl oligoribonucleotides to exclude

exon 23 and restore the dystrophin reading frame.111 This result

was recapitulated in vivo by intramuscular injection in mdx,

which showed efficient AO nuclear uptake and sarcolemmal

localization of dystrophin in treated muscle fibers.112 As a

result, two different AON chemistries have been under extensive

study for clinical application: 2′-O-methyl-phosphorothioate

(2′OMePS) and phosphorodiamidate morpholino oligomers

(PMOs). Both 2′OMePS and PMO-induced exon-skipping

approaches have been evaluated in cultured muscle cells from

DMD patients, GRMD/CXMDJ dogs, and H2K-mdx and

human explants (reviewed in Arechavala-Gomeza et al).113

Further, systemic delivery of exon 23–skipping antisense

compounds in mdx has been successful in restoring up to 50%

of dystrophin expression in various muscle groups, improved

muscle force, and reduced CK levels without tissue toxicity.114

Specific exon 51 skipping was established using intramuscular

injection of PMOs in mdx52115 and for human exons 44, 46,

and 49 by 2′OMePS in hDMD mice,30 which restored dystro-

phin expression in whole-body skeletal muscles in addition to

improving muscle function.30,115 Use of multiexon-skipping

“cocktails” in vivo was first achieved by systemic PMO delivery

in CXMDJ,47 and subsequently validated using 2′OMePS and

PMO combinations in CXMDJ116 and mdx4cv.117 This approach

appears more successful in increasing dystrophin expression in

CXMDJ dogs (26% of normal levels)116 compared to mdx4cv

(5%–7%)117 and trigger improvement in whole-body canine

skeletal muscle (with the exception of heart) without adverse

effects.47 Further, PMOs generally illustrate in vivo superior-

ity to 2′OMePS in the consistent and sustained induction of

dystrophin protein.118

Encouragingly, 2′OMePS and PMO exon 51–skipping

technologies have progressed to clinical trials, with early

indications of success at the biochemical and clinical level.

Proof of concept for PRO-051, a 2′OMePS AON developed

by Prosensa,119 was established by intramuscular injection

into DMD patients, which restored local sarcolemmal dystro-

phin in 64%–97% dystrophin-positive fibers and expression

between 17% and 35% with no adverse effects.120 This result

is impressive, considering that a dystrophin expression level

of 30% is postulated to avoid human pathogenesis,121 although

the precise level required to induce clinical and functional

improvement remains unclear.75 A follow-up Phase I/IIa

clinical trial using systemic administration of PRO-051 was

also well tolerated, with dose-dependent molecular efficacy

(60%–100% positive fibers and up to 15.6% expression)

accompanied by modest clinical improvement after 12

weeks’ extended treatment.122 PRO-051 is currently licensed

by GlaxoSmithKline (GSK2402968), who have initiated

three clinical trials, including a large international Phase III

study.119 Prosensa have also opened a Phase I/IIa clinical study

of PRO-044 (targeting exon 44) after assessment in DMD

cultured cells, with preclinical trials of PRO-045 and PRO-

053 (targeting exons 45 and 53, respectively) planned.119

AVI-4658 (Eteplirsen) is a splice-switching PMO

developed by Sarepta Therapeutics,123 identified by exon

51–specific AON screening in two different chemical forms

in cultured human muscle cells and explants (wild type

and DMD) and by local in vivo administration in hDMD

mice.124 AVI-4658 has also been tested in cultured myoblasts

of the CKCS-MD dog, which restored the reading frame

and protein.44 A single-blind, placebo-controlled, dose-

escalation study illustrated encouraging local dystrophin

induction,125 leading to a systemic intravenous Phase IIb

dose-escalation study to assess further the safety, tolerability,


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and pharmacokinetic properties of AVI-4658.126 The initial

12-week study proved disappointing compared to PRO051,

with 0%–55% positive fibers and up to 18% expression, with

no significant improvement in clinical outcomes (even at

higher doses), despite restoration of both components of the

DAPC and neural nitric oxide synthase (nNOS) to the sar-

colemma.126 Although a subsequently longer clinical regime

(24 weeks) has recently purported to improve dystrophin

induction (averaging 22.5% dystrophin-positive fibers),123

data on other parameters are currently unavailable. It is

likely that additional trials to reoptimize delivery and dos-

age of AVI-4658 are planned by Sarepta Therapeutics, who

have several other exon-skipping candidates, in particular

AVI-5038 (targeting exon 50), which is currently in preclini-

cal development.123

An alternative exon-skipping methodology involves

masking splicing sites using the endogenous targeting

capacity of modified small nuclear RNAs (snRNAs), in

particular U7 snRNA, to shuttle coupled AONs after rAAV

vectorization (reviewed in Benchaouir and Goyenvalle).110

Proof of principle was established in exons 48–50 deleted

DMD patient cell lines, where U1/U7 snRNA successfully

altered dystrophin pre-mRNA splicing to rescue synthesis,127

confirmed by exon 23 skipping in mouse C2C12 cells.128

In vivo systemic rescue of mdx dystrophic muscle by

single intravenous (IV) administration of exon 23–targeted

rAAV-U7 constructs induced sustained muscle expression

and correction of dystrophic pathology;129 parameters con-

firmed in rAAV-U1 and -U7 transduced mdx muscle after

local injection.130 The remarkable potential of systemic

IV rAAV-U7–mediated therapeutics follows recent, single

treatment of self-complementary rAAV-U7–mediated exon

skipping in dko, which restored near-normal dystrophin

levels and improved function in all muscles examined,

including heart.131 Human-specific multiexon skipping has

also been achieved using rAAV-U7 in DMD cell lines and

hDMD mice.132 Combined with the recent success of using

rAAV-U7–mediated exon 7 skipping in long-term restora-

tion of dystrophin expression in GRMD cardiac muscle,133,134

this approach illustrates significant potential in effectively

targeting DMD cardiomyopathy.

Increasing emergence of proof-of-principle studies in

gene-based dystrophin replacement and endogenous aug-

mentation provide significant promise for treatment of DMD

pathology, including the recent use of meganucleases and

zinc-finger nucleases to induce endogenous microdeletion

or -insertions in the endogenous gene.135 Despite the lack of

long-term toxicology studies, multiple AON-mediated exon

skipping potentially provides an applied therapeutic strategy

for up to 83% of DMD patients.136 However, difficulties in

establishing long-term correction and circumventing immune

challenges remain problematic, especially the inability

of gene-replacement and PMO/2′OMePS-mediated exon

skipping to effectively target cardiac tissue in mdx at doses

corresponding to those required for clinical application.137,138

Several other issues, such as the timing of repeated admin-

istration, optimization of systemic delivery, and addressing

poor cellular uptake, represent major hurdles in alleviating

numerous chemical, clinical, and ethical issues. Moreover,

further studies are required to clarify the mechanism through

which AONs interfere with RNA splicing to optimize target

sequences in humans.139,140 Recent studies to address these

issues link inhibition of cell-cycle progression to enhance

exon skipping,141 exonic sequences as better exon-skipping

targets, and enchanced efficacy by repeated intraperitoneal

delivery over intramuscular or IV injection.137 Encouragingly,

significant progress has been made in improving systemic

delivery (especially in cardiac muscle) and lowering dosage

of AONs in a number of animal models (including mdx, dko,

and GRMD) by conjugation to nanoparticles, cell-penetrating

peptides, or enhanced delivery using artificial vesicles

(reviewed in Arechavala-Gomeza et al113 and Moulton).142 It

is therefore likely the first definitive DMD therapy will result

from combining optimized multiexon-skipping methodolo-

gies with developing cell and pharmacological approaches.

Pharmacological approachesDMD pharmacotherapy strategies involve the systemic deliv-

ery of small compounds that aim to (1) provide sarcolemmal-

based compensation to directly address loss of the DAPC or

(2) modify dysfunctional signaling pathways implicated in

secondary pathology (Figure 1). A number of pharmacologi-

cal strategies show efficacy in circumventing immunological

and delivery hurdles that currently hamper gene- and cell-

based therapies.143 However, as pharmaceuticals frequently

target molecules involved in complex signaling pathways,

their development is far from simple. Here, we summarize

pharmacological approaches according to their intended

molecular targets and discuss their progress, pitfalls and

promise as treatment strategies for DMD.

Targeting primary DMD pathology by functional compensation or restoration of the DAPCIn conjunction with medicinal chemistry, a number

of pharmacological approaches have been devised to


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specifically address the primary defect in DMD. These

include (1) specific restoration of the DAPC by suppressing

nonsense mutations in the dystrophin gene; (2) upregulation

of its autosomal paralogue, utrophin, to provide a scaffold

on which components of the DAPC can be restored

to the sarcolemma; or (3) compensatory formation of

integrin–laminin complexes, which have mechanosignaling

similarities to the DAPC.

Aminoglycoside-mediated suppression of nonsense mutationsApproximately 10%–15% of DMD mutations convert an

amino acid into a premature stop codon, while the rest of

the mRNA is unaffected.144 Nonsense mutation read-through

strategies use aminoglycosides or small molecules to modify

ribosomes to produce full-length functional protein by spe-

cifically targeting premature stop codons through contextual

recognition of surrounding nucleotide sequences that differ

between nonsense mutations and regular stop codons.145 The

most studied aminoglycoside for DMD therapeutic use is

gentamicin, which acts via the 40S ribosomal subunit.146

Proof-of-concept studies of gentamicin in mdx have been

promising: a 2-week course of subcutaneous injection suc-

cessfully enabled full-length dystrophin production with

correct localization properties in both skeletal muscle147

and the vascular system.148 Further, these studies indicated

improvement in a number of physiological parameters,

including protection against contractile injury, normaliza-

tion of blood flow and increased cardiac response to sheer

stress.147,148 However, following Phase I DMD/BMD clini-

cal outcomes were highly variable, from promising143,149 to

disappointing.150 Potency issues due to batch consistency151

or dosage regimes may contribute to such conflicting out-

comes, although the latter is less likely, given follow-up

mdx studies could not replicate benefits described in one

clinical trial.152 In an attempt to combat gentamicin toxic-

ity and increase target specificity, a drug-delivery system

using hybrid liposomes has been developed,153 and it will

be of interest if this approach results in future clinical

assessment.

The small-molecule ataluren (PTC124), which also acts

via the 60S ribosomal subunit,154 exhibits similar efficiency

in mdx to gentamicin at a lower concentration.155 Although

PTC124 was well tolerated in patients, three Phase II DMD/

BMD clinical studies were halted as predetermined primary

outcomes were not met.69 Therefore, despite the favorable

pharmacodynamic response of both gentamicin and ataluren,

their clinical development remains problematic, making their

path to regulatory approval for DMD therapy a difficult one.143

To circumvent toxicity concerns, an alternative approach

is use of less toxic antibiotic peptides, such as negamycin,

which inhibits eukaryotic RNA decoding.156 Encouragingly,

prolonged (4-week) intraperitoneal delivery in mdx enabled

restoration of cardiac and skeletal muscle dystrophin levels

comparable to those achieved with gentamicin,156 making

negamycin a promising therapeutic candidate.

Utrophin upregulationA compensatory approach aimed at restoring components

of the DAPC involves increasing levels of utrophin, the

autosomal paralogue of dystrophin.157 Although spatially

restricted in adult myof ibers to neuromuscular and

myotendinous junctions,158 extrajunctional utrophin is

upregulated during embryonic development in mdx and

DMD patients.159 Utrophin-based upregulation therapy has

a number of favorable attributes, notably (1) the ability to

circumvent immunological challenges that accompany intro-

duction of functional dystrophin protein; (2) in principle,

effectiveness for all DMD patients, regardless of gene defect;

and (3) amenability to systemic administration, given whole-

body overexpression in mdx appears nontoxic.160 Extensive

proof-of-principle studies in mdx establish that a three- to

fourfold increase in utrophin expression can enable functional

restoration by formation of an alternative to the DAPC:

the utrophin-associated protein complex (UAPC) complex

(reviewed in Moorwood et al).161 Historically, a number of

endogenous transcriptional/posttranscriptional effectors of

utrophin have been evaluated in mdx, including direct injec-

tion of the active Ras homologue gene family, member A

(RhoA), heregulin, NO, and l-arginine, but none of these

approaches has been able to reproducibly increase utrophin

levels (extensively reviewed in Fairclough et al).162

The observation that endogenous utrophin is elevated

in slow-twitch muscle163 led to investigation of how key

regulators of muscle oxidative metabolism can be aug-

mented to obtain therapeutic levels of utrophin. Targeted

upregulation of either peroxisome proliferator–activated

receptor cofactor 1-alpha (PGC-1α),164,165 its downstream

effector peroxisome proliferator–activated receptor beta/

delta (PPARβ/δ), GA-binding protein (GABP) α/β, active

calcineurin (CnA*), or associated nuclear factors of activated

T cells (NFAT) in mdx mice illustrate a twofold increase

in utrophin mRNA levels (reviewed in Fairclough et al).162

Encouragingly, many of these targets illustrate promoter-

based synergism,166,167 indicating a multitargeting utrophin

approach is feasible. However, it is important to note that, at


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present, pharmacological optimization of individual targets

holds varying promise. For example, the biological benefit of

activating PPARβ/δ using the histone deacetylase inhibitor

valproic acid (VPA) and its derivatives may be outweighed

by concerns over their developmental toxicity.168 Similarly,

synthetic PPAR ligands are under evaluation for non-DMD

therapies,169 but complications including off-target kinase

activation170 and severe side effects have led in some cases

to recall and reformulation.171 A more promising approach

is administration of the adenosine monophosphate analog

5-aminoimidazole-4-carboxamide ribotide (AICAR). AICAR

activates PGC-1α and PPARβ/δ via AMP-activated protein

kinase (AMPK),172 potentially affording greater therapeutic

effect by enhancing synergism between PGC-1α with the α

subunit of GABP.166 Encouragingly, AICAR administration

elevates sarcolemmal utrophin and β-DG protein levels

and fast-to-slow muscle-fiber transition173 in mdx similar to

using PPARβ/δ agonist GW501516167 and AAV-mediated

PGC-1α delivery.164,165 As AICAR is in frequent clinical

use,174 its future clinical assessment in DMD patients thus

seems likely.

An alternate promoter-based utrophin strategy involves

artificial zinc-finger proteins fused with effector domains

(ZF-ATF), which is currently being evaluated in mdx (see

Passananti et al).175 However, the current favored strategy is

identification of orally deliverable compounds with utrophin

upregulation capabilities (reviewed in Moorwood et al).161

Proof of principle was established by functional screening

of chemical scaffold candidates, resulting in optimization of

an orally bioavailable 2-arylbenzoxazole derivative – SMT-

C1100 (BMN195).176 Daily SMT-C1100 administration in

mdx improved membrane integrity and demonstrated syn-

ergism with prednisolone.176 However, a move into Phase I

safety trials in healthy individuals was discontinued due to

insufficient levels of SMT C1100 in plasma,177 a difficulty

being addressed by reformulation. However, the lack of

safety issues with SMT-C1100 is encouraging, evidenced

by complementary studies using compound libraries of

FDA-approved and natural substances,161 pharmacological

interest (Zalicus and PTC Therapeutics),154 and development

of improved screening assays.178

Protein-based therapy: TAT-utrophin and biglycanDirect protein replacement of utrophin in dystrophin-

deficient muscle uses deliverable chimeras constructed by

fusing the transactivator of transcription (TAT) protein trans-

duction domain (PTD) of human immunodeficiency virus

(HIV-1)179 with micro-utrophin (mUtr) protein (TAT-mUtr).180

Intraperitoneal injection of TAT-mUtr in mdx established

functional sarcolemmal mUtr–glycoprotein complexes,

leading to improved membrane integrity and contractile

function.180 As similar levels of functional improvement in

dko mice establish this approach as an attractive therapeu-

tic possibility,181 rigorous optimization is being performed

prior to preclinical safety and toxicology studies.182 Pending

results, clinical TAT-mUtr trials (Retrophin, compound

RE-001)182 are anticipated in late 2012.

A related protein-based pharmacological candidate is

biglycan, a small leucine-rich proteoglycan found at elevated

levels within the ECM of DMD patient skeletal muscle.183

Biglycan is a critical regulator of sarcolemmal proteins

such as nNOS, components of the DAPC, and utrophin in

particular, during the muscular response to cell damage and

apoptosis.184 Single systemic administration of recombinant

human biglycan (rhBGN) was well tolerated and sufficient to

counteract mdx pathology by enhancing UAPC stabilisation,

as an identical dose regime was ineffective in dko mice.185

To mitigate off-target effects, active rhBGN is currently

being manufactured without biglycan-associated complex

carbohydrate side chains for preclinical evaluation (Tivorsan,

compound TVN-102).183

α7-integrin upregulation/laminin-111Integrin/laminin complexes act as mechanosignaling anchors,

linking ECM laminin and fibronectin with intercellular

cytoskeletal components.186 Similar to the structural and

signaling role of the DAPC, α7β1-integrin/laminin-211 com-

plexes act as crucial enablers of muscle development, repair,

regeneration, and integrity in skeletal muscle.187,188 Indeed the

degree of functional redundancy between integrin/laminin

complexes and the DAPC coupled with endogenous eleva-

tion of sarcolemmal α7β1 protein in DMD patients and mdx

mice indicated that α7-integrin upregulation may stem DMD

muscle pathology.187 Efficacy of functional compensation

was established by transgenic overexpression of α7-integrin

in dko mice, which was effective in extending longevity

(threefold), reducing kyphosis and increasing mobility as a

result of increased sarcolemmal α7β1 protein.189 While not

preventing initial degeneration, α7-integrin upregulation

appears to mediate sarcolemmal stability after subsequent

regeneration by promoting SC proliferation, adherence,

and activation.189,190 Favorably, inducing α7-integrin over-

expression does not demonstrate visible toxicity or affect

in vivo global gene-expression profiles,190 and, as a result,

small-compound screening for α7-integrin upregulators has

been initiated.


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Similar to utrophin, compound screening for α7-integrin

provides a relatively uncomplicated means to develop orally

bioavailable molecules to complement other DMD therapies

or benefit patients ineligible for strategies such as exon

skipping. VPA was identified as an α7-integrin upregula-

tion compound using a cell-based assay, and intraperitoneal

injection of VPA in dko mice results in decreased fibrosis,

hypertrophy, and increases sarcolemmal integrity.191 However,

contrary to observations from in vitro studies, α7-integrin

levels remain unchanged.191 This discrepancy may be

explained by the observation that VPA and α7-integrin both

act, albeit independently, via the acutely transforming retro-

virus AKT8 in rodent T-cell lymphoma (Akt)/ mammalian

target of rapamycin (mTOR) signaling pathway191 to

positively regulate skeletal muscle hypertrophy.192 Thus,

in vivo VPA administration alone appears sufficient to trig-

ger Akt- mediated signaling independently of α7-integrin.191

However, as previously outlined in the utrophin section, the

potential toxicity of VPA required to achieve clinical benefit

remains a concern.168

An alternative candidate identified via small-compound

screening is laminin-111 (LAM-111).193 Intramuscular or

systemic LAM-III injection in mdx has enabled the suf-

ficient induction of α7-integrin to achieve both sarcolem-

mal stabilisation193 and increased regenerative capacity.194

Although this study has been countered by the failure of

enhancing heterodimer LAM-111 formation in improving

dystrophic skeletal muscle morphology in mdx mice,195 the

use of validated α7-integrin effectors has clinical promise.

To preclude compounds with toxicity concerns, current

pharmacological strategies are based on FDA-approved

drug libraries (Prothelia)196 or synergistic approved drug

approaches (Zalicus).197 Further, a number of lead com-

pounds are currently at the preclinical (LAM-111/PRT-01

and PRT-20) and discovery (PRT-300) stages.196

Targeting secondary DMD pathology resulting from dystrophin deficiencyAlthough the pathological presentation of dystrophin

deficiency has been traditionally classified according to

phenotypical and biochemical parameters such as fibrosis,

necrosis, oxidative stress, and inflammation, it is increas-

ingly apparent that the molecular processes that underlie

these processes are intimately linked.198 As a result, phar-

macological intervention devised to alleviate one of these

parameters may result in either assisting or even hindering

one another. This is apparent with long-term corticosteroid

treatment, which is thought to act by positively modifying

both inflammation and Ca2+ homeostasis.21 With this in mind,

we categorize the progression of pharmacological interven-

tion approaches according to their original aim of targeting

a specific pathological or cellular defect, while outlining

their links to others.

Reactive oxygen species and intracellular Ca2+ influxFragility of the DAPC leads to stretch-induced membrane

permeability and Ca2+ influx, which activates proteases and

enhances mitochondrial production of ROS,17 which in turn

regulates the nuclear factor kappa-light-chain enhancer of

activated B cells (NF-κB) complex.199 Although the synthetic

membrane sealant polyaxamer 188 (PA188) shows promise

in reducing membrane permeability in mdx200 and GRMD

models,201 the primary strategy to aid sarcolemmal integrity

involves supplementing the antioxidant response in DMD

patients to normalize redox balance and protect against oxida-

tive stress (reviewed in Tidball and Wehling-Henricks).202

Dietary supplementation using glutamine, which is nor-

mally depleted in DMD patients,203 prevents glucocorticoid-

mediated upregulation of the transforming growth factor beta

(TGF-β) family member myostatin (see section on fibrosis)204

and protects against oxidative stress in mdx.205 However, sim-

ilar to combinatorial approaches using creatine and alanine,

reproducibility in clinical trials is modest at best (reviewed in

Fairclough et al).162 In contrast, the commercial antioxidant

melatonin improves muscle redox status and reduces inflam-

mation in mdx5cv206 and DMD patients,207 while prenatal

administration of epigallocatechin gallate effectively reduces

ROS-mediated NF-κB activation in mdx208,209 to the extent

that Phase I/II DMD clinical trials are planned.210

Another promising supplement is N-acetylcysteine

(NAC), a direct ROS scavenger and indirect l-cysteine

precursor.211 NAC treatment in postnecrotic mdx-decreased

nuclear NF-κB protine enhances sarcolemmal UAPC forma-

tion, reduces stretch-induced damage,212 and protects against

damage and necrosis after only a week of treatment.213

Further, linking antioxidants to lipophilic cations has human

efficacy and improves effectiveness of antioxidants in dis-

ease models.214 A recent study directly addressed calcium

influx in mdx and dko muscle via oral gavage of BGP-15

(O-[3-piperidino-2-hydroxy-1-propyl]nicotinic amidoxime),

increasing expression of heat shock protein 72, which binds

and preserves sarcoplasmic reticulum Ca2+-ATPase SERCA

under cellular stress.215 As BGP-15 administration has been

effective in slowing progression, preserving strength, and

improving muscle function in dko,215 this methodology holds

immense therapeutic potential for DMD.


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Mitochondrial permeability transition poreSustained increase of [Ca2+]i and the resultant redox imbal-

ance in dystrophic muscle induces formation of the mito-

chondrial permeability transition pore (MPTP), leading

to a self-perpetuating cycle of ROS, TNF-α, and NF-κB

and eventual cell death (reviewed in Lemasters et al).216

Attempts to desensitize mitochondria using cyclosporine

A (CsA)-mediated blockage of cyclophilin D217,218 in mdx

decreased necrosis in some studies219 but not others.220 This

result was reflected in DMD clinical trials where a small

study reported improvements in muscle-force generation,221

but a following, larger randomized trial illustrated little

benefit.222 To circumvent the effect of long-term CsA treat-

ment on both the immune system and calcineurin signaling,

use of a nonimmunosuppressing CsA analog (Debio-025)

was recently shown to enhance MPTP blockade over CsA

or prednisolone in mdx, and is under assessment for other

muscular dystrophies (reviewed in Fairclough et al).162

NecrosisDysregulation of the NO/cyclic guanosine monophosphate

(cGMP) pathway has been implicated in the loss of contractile

performance and sarcolemmal integrity in dystrophic mus-

cle.223 Increased [Ca2+]i levels in dystrophic muscle leads to

increased catalysis of NO (a free radical scavenger and regu-

lator of the NO/cGMP signaling pathway) from l-arginine

by NOS, whereas limiting [Ca2+]i initiates NO-facilitated

relaxation.224 Further, loss of the DAPC in mdx and DMD

muscle prevents anchoring of nNOS at the sarcolemma,

which also decreases NO levels and results in myofiber

damage.10 Conversely, transgenic NOS restoration in mdx

reduces ROS-mediated activation, decreases muscle damage/

fibrosis and enhances formation of UAPC complexes,225 and

NO-releasing agents such as the nonsteroidal anti-inflam-

matory drug HCT-1026 can prevent mdx muscle inflamma-

tion and damage to a greater extent than prednisolone.226 In

addition, several NO donors including MyoNovin, isorbide

dinitrate, or the analog guaifenesin dinitrate227 act to alleviate

multiple aspects of mdx pathology by activation of SCs224

and/or via alleviating glucocorticoid side effects.228 Similar

benefits are observed in mdx when catalysis of GMP to cGMP

is either increased by transgenic means223 or by using phos-

phodiesterase 5 (PDE5) inhibitors to prevent degradation of

cGMP.229 Further, PDE5 inhibitors deactivate hypertrophy

signaling pathways triggered by pressure load, including

those deregulated in DMD, such as calcineurin/NFAT,

phosphoinositide-3 kinase/Akt, and extracellular signal–

regulated kinase 1/2 cascades.229 Given that commercially

available cGMP-specific PDE5 inhibitors can prevent

and even reverse contraction-induced myofiber damage

(tadalafil)230 or cardiac hypertrophy (sildenafil)231 in mdx

mice, both are at the recruitment stage for DMD clinical

trials.232

Protein-degradation inhibitorsCysteine calpains are also activated in response to [Ca2+]i

in DMD,233 including the muscle-specific isoform absent in

limb-girdle muscular dystrophy-2A.234 Interestingly, calpain

modulation in dystrophic muscle primarily disrupts regula-

tory mechanisms influenced by calpains rather than increase

proteolytic activity.234 Alleviating mdx muscle degeneration

and necrosis by targetting calpain overactivity has been

successful, using natural (calpastatin) or pharmacological

means, including leupeptin, prodrug BN82270, dystrypsin

(camostat mesylate) and enhanced-uptake cell-penetrating

alpha-keto-amide calpain inhibitors (reviewed in Fairclough

et al).162 However, despite links between calpain inhibition

and TGF-β downregulation,235 calpastatin overexpression236

or administration of leupeptin-carnitine conjugate C101 was

ineffective in mdx236 and GRMD237 models. Further, calpain

inhibitors are also endogenously countered via elevated

proteasome activity,164,236 which may preclude their pharma-

cological use. Alternatively, proteasomal targeting by sys-

temic administration of nonspecific (MG-132) and specific

(bortezomib) ubiquitin ligase inhibitors effectively reduces

NF-κB–mediated inflammation and restores DAPC compo-

nents to the sarcolemma in mdx238–240 and DMD explants.241

Therefore, their synergistic use with compounds that restore

redox balance/provide functional compensation is possible

if a balance between benefit and side effects from long-term

use can be achieved.

InflammationPrior to the onset of visible muscle damage, increased

TNF-α leads to induction of IκB kinase (IKK) mediated

NF-κB signaling,242 a major contributor to the inflammatory

and necrotic response of DMD myofibers.243 Indeed, NF-κB

activation leads to aberrant signaling inexorably linked to

increased ROS, and this synergism significantly contrib-

utes to the preliminary wave of inflammation in secondary

DMD pathology.244 Further, glucocorticoids exert positive

effects through NF-κB inhibition,245 indicating that specific

pharmacological targeting of NF-κB may have therapeutic

benefit.

Direct TNF-α inhibition using the anti–TNF-α antibody

infliximab, or depletion of circulating TNF-α levels using


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receptor decoy protein (etanercept) decrease fibrosis and

necrosis and improve muscle function in mdx (reviewed in

Fairclough et al).162 Blocking downstream targets of NF-κB

signaling such as cyclooxygenase-2 (COX-2) by curcumin

improves sarcolemmal integrity and muscle strength in mdx,

in addition to decreasing CK and levels of factors involved in

the inflammatory process, including TNF-α and NF-κB.246,247

Unfortunately, the progressive increase of NF-κB levels

in dystrophic muscle become increasingly resistant to

curcumin,247 indicating that targeted COX-2 inhibition may

not provide benefit in a clinical setting. Nevertheless, a

DMD Phase I safety trial is planned for the COX-2 inhibi-

tor Flavocoxid.69 In contrast to curcumin, direct targeting

of IKK using rAAV-mediated intramuscular administration

of dominant negative IKK protein improves regeneration

in older but not younger mdx mice.248 Impressively, sys-

temic delivery of IKK inhibitory peptide (NF-κB essential

modulator binding domain [NBD]) increases regeneration,

reduces necrosis, and improves contractile function in mdx

and dko diaphragm.249 Optimizing intracellular delivery of

NBD by fusion with a cationic cell–penetrating octalysine

peptide250 (8K-NBD) leads to further improvement in mdx

histology.251 Further, the ability of 8K-NBD to enhance

benefits provided by AAV9-mDYS delivery252 indicates

that IKK-mediated NF-κB inhibition may assist in treating

residual fibrosis and necrosis observed with gene-transfer

approaches.

FibrosisPathological f ibrosis in DMD muscle correlates with

increased TGF-β signaling,254 which hallmarks increased

type I collagen production253 and the upregulation of

several key intracellular markers of inflammation and

oxidative stress.253 TGF-β antagonists suramin and deco-

rin have shown efficacy in promoting muscle recovery by

attenuating sarcolemmal damage, decreasing fibrosis, and

enhancing muscle regeneration (reviewed in Burks and

Cohn).255 Oral administration of nontoxic antifibrotics such

as Bowman–Birk inhibitor and imatinib inhibit upstream

and downstream TGF-β effectors, respectively, to affect a

phenotype similar to direct antagonists in mdx.256–258 The

plant alkaloid halofuginone (granted orphan drug status

for DMD as HT-100)259 acts as a potent inhibitor of TGF-β

profibrotic signaling to recapitulate these parameters in mdx

by enhancing myotube fusion259 and function in initial260 and

established fibrosis,261 negating the necessity for accurate

therapeutic timing. It is also interesting to note that the

TNF-α receptor decoy etanercept (see previous section)

also reduces type I collagen and TGF-β mRNA,262 indicating

TNF-α blockade approaches may be effective in modulating

TGF-β–mediated fibrosis.

TGF-β signaling can also be indirectly mediated by

blocking bone morphogenic protein (BMP) ligands or the

renin–angiotensin system (RAS), as both are continuously

elevated in mdx and DMD skeletal muscle (reviewed in Burks

and Cohn).255 BMP antagonists noggin, dorsomorphin, and

LDN-193189 enhance differentiation in human myoblasts263

and intramuscular AAV delivery of the most potent and

selective antagonist, noggin (“ad-noggin”), in dko mice

enhances regeneration and alleviated dystrophic patholo-

gy.263 However, repressing BMP signaling may influence

toxicity and severity of side effects, parameters that preclude

long-term in vivo administration of dorsomorphin.263 RAS

inhibition is considered a recent promising approach, where

administration of angiotensin converting enzyme inhibi-

tors (ACEi), antagonists of the angiotensin II (ATII type I)

receptor, or TGF-β–neutralising antibodies (such as ID-11)

demonstrate improvements in mdx pathology such as reduced

fibrosis, increased muscle strength, and enhanced respiratory

function.264–266 Further, ATII receptor agonists may counter

effects mediated by NF-κB, such as inflammation- and oxida-

tive stress–related muscle damage.267 As early intervention

using combined ACEi/ATII type I antagonists preserves

muscle function in dko to an extent currently unparalleled

by other pharmacological strategies,267 preclinical evaluation

is highly anticipated.

Blocking secreted myostatin,268 a TGF-β–related nega-

tive regulator of muscle growth,269 also increases muscle-

fiber size. Myostatin-null mice illustrate robust muscular

hypertrophy and hyperplasia by deregulation of myoblast

proliferation and differentiation.268 Similar improvements

in mdx have been achieved using neutralizing antibodies,

myostatin propeptide (MRPO) follistatin-derived pep-

tides and the soluble extracellular form of the myostatin

activin type-II receptor (reviewed in Burks and Cohn).255

Unfortunately, Phase II trials of recombinant ActRIIB

decoy (ACE-031) were suspended due to safety issues,270

and PhaseI/II clinical trials of antimyostatin MYO-029

antibody (stamulumab), although well tolerated, did not

improve muscle strength.271 These findings impact alternate

approaches, as clinical trials are not planned for AAV8-

mediated MRPO delivery validated in the GRMD model.272

However, myostatin-blockade approaches have benefited

from exon-skipping methodologies developed for dystro-


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phin, where induction of mdx muscle hypertrophy using

destructive 2OMP/PMO myostatin pre-mRNA targeting273

has advanced to 2′OMePS-based strategies simultaneously

targeting myostatin and dystrophin.274

Muscle-growth strategies also involve exogenous deliv-

ery of insulin-like growth factor I (IGF-I), which stimulates

SC proliferation and differentiation during muscle regen-

eration.275 Subcutaneous injection or viral expression of

human IGF-I (rhIGF-I) or polyethylene glycol-modified

IGF-I analogs (PEG-IGF-I) in mdx increase muscle strength

and resistance to fatigue,276,277 but are ineffective against

mechanical injury and myofiber degeneration.278 Further,

PEG-IGF-I administration in dko and older mdx mice high-

light somewhat limited potential to ameliorate severe or

established pathophysiology, and the authors suggest deliv-

ery should be initiated only for mild muscle pathologies.277

Nevertheless, an IGF-I Phase I clinical trial is currently at

the recruitment stage.69

ConclusionIn recent years, significant progress has been made in the

discovery of novel DMD therapeutic strategies and the

continued development of established gene- and cell-based

protocols. This is due, in part, to the continued understanding

of molecular mechanisms that underlie DMD pathogenesis

and the ability to establish efficacy using an increasing array

of animal models. The development of a definitive DMD

therapy is increasingly likely to involve synergism between

adjunctive pharmaceuticals with gene-based approaches

(such as exon skipping) to target multiple aspects of dystro-

phic pathology. Further, advances in cell-based technology

show distinct promise in aiding efforts to correct endogenous

dystrophin by their ability to act as autologous delivery

vehicles. The successful move to clinical trials in each field

has not only highlighted important aspects in the treatment

and management of DMD but has also provided useful

information for future design to accurately determine the age

and state of the disease where treatment has clinically mean-

ingful benefit. It is also increasingly apparent that accurate

genetic diagnosis is key, given the increasing development

of mutation-specific molecular therapies. As outlined in this

review, many challenges lie ahead in the development and

delivery of DMD therapeutics, and the specific approach(es)

that will eventually result in success is unclear. However, it

is clear that despite various hurdles, the incredible progress

in therapeutic design in recent years has led to improved

methodologies with immense translational potential.

DisclosureKED is a consultant for Summit Plc. and is on the Scientific

Advisory Board of Prosensa Plc. The authors report no

other conflicts of interest in this work.

References 1. Dubowitz V. Muscle disorders in childhood. Major Probl Clin Pediatr.

1978;16:iii–xiii, 1–282. 2. Emery A. Muscular dystrophy – the facts. Neuromuscul Disord. 1995;

5(6):521. 3. Bushby K, Finkel R, Birnkrant DJ, et al. Diagnosis and management of

Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010;9(1):77–93.

4. Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature. 1986;323(6089):646–650.

5. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6): 919–928.

6. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987;50(3):509–517.

7. Levine BA, Moir AJ, Patchell VB, Perry SV. The interaction of actin with dystrophin. FEBS Lett. 1990;263(1):159–162.

8. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992;355(6362):696–702.

9. Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol. 2006;7(10):762–773.

10. Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide syn-thase complexed with dystrophin and absent from skeletal muscle sarco-lemma in Duchenne muscular dystrophy. Cell. 1995;82(5):743–752.

11. Sadoulet-Puccio HM, Rajala M, Kunkel LM. Dystrobrevin and dys-trophin: an interaction through coiled-coil motifs. Proc Natl Acad Sci U S A. 1997;94(23):12413–12418.

12. Yang B, Jung D, Rafael JA, Chamberlain JS, Campbell KP. Identification of alpha-syntrophin binding to syntrophin triplet, dystrophin, and utrophin. J Biol Chem. 1995;270(10):4975–4978.

13. Lynch GS, Rafael JA, Chamberlain JS, Faulkner JA. Contraction-induced injury to single permeabilized muscle fibers from mdx, trans-genic mdx, and control mice. Am J Physiol Cell Physiol. 2000;279(4): C1290–C1294.

14. Abdel-Salam E, Abdel-Meguid I, Korraa SS. Markers of degenera-tion and regeneration in Duchenne muscular dystrophy. Acta Myol. 2009;28(3):94–100.

15. Foidart M, Foidart JM, Engel WK. Collagen localization in normal and fibrotic human skeletal muscle. Arch Neurol. 1981;38(3):152–157.

16. Morandi L, Mora M, Gussoni E, Tedeschi S, Cornelio F. Dystrophin analysis in Duchenne and Becker muscular dystrophy carriers: correlation with intracellular calcium and albumin. Ann Neurol. 1990;28(5):674–679.

17. Nethery D, Callahan LA, Stofan D, Mattera R, DiMarco A, Supinski G. PLA(2) dependence of diaphragm mitochondrial formation of reactive oxygen species. J Appl Physiol. 2000;89(1):72–80.

18. McDouall RM, Dunn MJ, Dubowitz V. Expression of class I and class II MHC antigens in neuromuscular diseases. J Neurol Sci. 1989; 89(2–3):213–226.

19. De Paepe B, Creus KK, Martin JJ, De Bleecker JL. Upregulation of chemokines and their receptors in Duchenne muscular dystrophy: potential for attenuation of myofiber necrosis. Muscle Nerve. Epub Jun 4, 2012.


Dovepress

Dovepress

157


www.dovepress.com

www.dovepress.com

www.dovepress.com


20. Ichim TE, Alexandrescu DT, Solano F, et al. Mesenchymal stem cells as anti-inflammatories: implications for treatment of Duchenne muscular dystrophy. Cell Immunol. 2010;260(2):75–82.

21. DeSilva S, Drachman DB, Mellits D, Kuncl RW. Prednisone treatment in Duchenne muscular dystrophy. Long-term benefit. Arch Neurol. 1987;44(8):818–822.

22. Moxley RT 3rd, Ashwal S, Pandya S, et al. Practice parameter: corticosteroid treatment of Duchenne dystrophy: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society. Neurology. 2005;64(1):13–20.

23. Willmann R, Possekel S, Dubach-Powell J, Meier T, Ruegg MA. Mammalian animal models for Duchenne muscular dystrophy. Neuromuscul Disord. 2009;19(4):241–249.

24. Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A. 1984;81(4):1189–1192.

25. Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989;244(4912):1578–1580.

26. Stedman HH, Sweeney HL, Shrager JB, et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature. 1991;352(6335):536–539.

27. Chapman VM, Miller DR, Armstrong D, Caskey CT. Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice. Proc Natl Acad Sci U S A. 1989;86(4):1292–1296.

28. Helderman-van den Enden AT, Straathof CS, Aartsma-Rus A, et al. Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients. Neuromuscul Disord. 2010;20(4):251–254.

29. Araki E, Nakamura K, Nakao K, et al. Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy. Biochem Biophys Res Commun. 1997;238(2):492–497.

30. Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ, et al. Targeted exon skipping in transgenic hDMD mice: a model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther. 2004;10(2):232–240.

31. Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev. 1996;10(10):1173–1183.

32. Grady RM, Grange RW, Lau KS, et al. Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies. Nat Cell Biol. 1999;1(4):215–220.

33. Raymackers JM, Debaix H, Colson-Van Schoor M, et al. Consequence of parvalbumin deficiency in the mdx mouse: histological, biochemi-cal and mechanical phenotype of a new double mutant. Neuromuscul Disord. 2003;13(5):376–387.

34. Guo C, Willem M, Werner A, et al. Absence of alpha 7 integrin in dystrophin-deficient mice causes a myopathy similar to Duchenne muscular dystrophy. Hum Mol Genet. 2006;15(6):989–998.

35. Chandrasekharan K, Yoon JH, Xu Y, et al. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2010;2(42):42ra54.

36. Deconinck AE, Rafael JA, Skinner JA, et al. Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell. 1997;90(4):717–727.

37. Zhou L, Rafael-Fortney JA, Huang P, et al. Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice. J Neurol Sci. 2008;264(1–2):106–111.

38. Shelton GD, Engvall E. Canine and feline models of human inherited muscle diseases. Neuromuscul Disord. 2005;15(2):127–138.

39. Sharp NJ, Kornegay JN, Van Camp SD, et al. An error in dystro-phin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics. 1992;13(1):115–121.

40. Shimatsu Y, Katagiri K, Furuta T, et al. Canine X-linked muscular dystrophy in Japan (CXMDJ). Exp Anim. 2003;52(2):93–97.

41. Winand N, Cooper B. Molecular characterization of severe Duchenne-type muscular dystrophy in a family of rottweiler dogs. In: Proceedings of Molecular Mechanisms of Neuromuscular Disease. Tucson: University of Arizona; 1994.

42. Schatzberg SJ, Olby NJ, Breen M, et al. Molecular analysis of a spontaneous dystrophin ‘knockout’ dog. Neuromuscul Disord. 1999; 9(5):289–295.

43. Jones BR, Brennan S, Mooney CT, et al. Muscular dystrophy with truncated dystrophin in a family of Japanese Spitz dogs. J Neurol Sci. 2004;217(2):143–149.

44. Walmsley GL, Arechavala-Gomeza V, Fernandez-Fuente M, et al. A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient cavalier king charles spaniels is amenable to exon 51 skipping. PLoS One. 2010;5(1):e8647.

45. Cooper BJ, Winand NJ, Stedman H, et al. The homologue of the Duch-enne locus is defective in X-linked muscular dystrophy of dogs. Nature. 1988;334(6178):154–156.

46. Shimatsu Y, Yoshimura M, Yuasa K, et al. Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ. Acta Myol. 2005;24(2):145–154.

47. Yokota T, Lu QL, Partridge T, et al. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann Neurol. 2009;65(6): 667–676.

48. Aartsma-Rus A, Janson AA, Kaman WE, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet. 2004;74(1):83–92.

49. Vos JH, van der Linde-Sipman JS, Goedegebuure SA. Dystrophy-like myopathy in the cat. J Comp Pathol. 1986;96(3):335–341.

50. Klymiuk N, Thirion C, Burkhardt K, et al. 238 tailored pig model of duchenne muscular dystrophy. Reprod Fertil Dev. 2011;24(1):231.

51. Winand NJ, Edwards M, Pradhan D, Berian CA, Cooper BJ. Deletion of the dystrophin muscle promoter in feline muscular dystrophy. Neuromuscul Disord. 1994;4(5–6):433–445.

52. Partridge T. Models of dystrophinopathy, pathological mechanisms and assessment of therapies. In: Brown SC, Lucy JA, editors. Dystrophin: Gene, Protein and Cell Biology. Cambridge: Cambridge University Press; 1997:310–321.

53. Meregalli M, Farini A, Colleoni F, Cassinelli L, Torrente Y. The role of stem cells in muscular dystrophies. Curr Gene Ther. 2012;12(3): 192–205.

54. Partridge TA, Grounds M, Sloper JC. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature. 1978;273(5660):306–308.

55. Mouly V, Aamiri A, Perie S, et al. Myoblast transfer therapy: is there any light at the end of the tunnel? Acta Myol. 2005;24(2):128–133.

56. Huard J, Roy R, Bouchard JP, Malouin F, Richards CL, Tremblay JP. Human myoblast transplantation between immunohistocompatible donors and recipients produces immune reactions. Transplant Proc. 1992;24(6):3049–3051.

57. Skuk D, Paradis M, Goulet M, Tremblay JP. Ischemic central necrosis in pockets of transplanted myoblasts in nonhuman primates: implications for cell-transplantation strategies. Transplantation. 2007;84(10):1307–1315.

58. Price FD, Kuroda K, Rudnicki MA. Stem cell based therapies to treat muscular dystrophy. Biochim Biophys Acta. 2007;1772(2): 272–283.

59. Jankowski RJ, Deasy BM, Huard J. Muscle-derived stem cells. Gene Ther. 2002;9(10):642–647.

60. Collins CA, Olsen I, Zammit PS, et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell. 2005;122(2):289–301.

61. Webster C, Blau HM. Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy. Somat Cell Mol Genet. 1990;16(6):557–565.


Dovepress

Dovepress

158

Perkins and Davies

www.dovepress.com

www.dovepress.com

www.dovepress.com


62. Torrente Y, Belicchi M, Marchesi C, et al. Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients. Cell Transplant. 2007;16(6):563–577.

63. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature. 1999;401(6751):390–394.

64. Mu X, Xiang G, Rathbone CR, et al. Slow-adhering stem cells derived from injured skeletal muscle have improved regenerative capacity. Am J Pathol. 2011;179(2):931–941.

65. Darabi R, Gehlbach K, Bachoo RM, et al. Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med. 2008;14(2):134–143.

66. Jackson WM, Nesti LJ, Tuan RS. Potential therapeutic applications of muscle-derived mesenchymal stem and progenitor cells. Expert Opin Biol Ther. 2010;10(4):505–517.

67. Ma X, Zhang S, Zhou J, et al. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo. J Tissue Eng Regen Med. 2012;6(8):598–613.

68. Kong KY, Ren J, Kraus M, Finklestein SP, Brown RH Jr. Human umbilical cord blood cells differentiate into muscle in sjl muscular dystrophy mice. Stem Cells. 2004;22(6):981–993.

69. ClinicalTrials.gov [homepage on the Internet]. Clinical Trials for Duchenne muscular dystrophy. Bethesda, MD: US National Library of Medicine; 2012 [cited August 8, 2012]. Available from: http://clinicaltrials.gov/ct2/results?term=dmd. Accessed August 18, 2012.

70. Sampaolesi M, Torrente Y, Innocenzi A, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science. 2003;301(5632):487–492.

71. Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science. 2005;309(5732):314–317.

72. Nitahara-Kasahara Y, Hayashita-Kinoh H, Ohshima-Hosoyama S, et al. Long-term engraftment of multipotent mesenchymal stromal cells that differentiate to form myogenic cells in dogs with Duchenne muscular dystrophy. Mol Ther. 2012;20(1):168–177.

73. Berry SE, Liu J, Chaney EJ, Kaufman SJ. Multipotential mesoangioblast stem cell therapy in the mdx/utrn-/- mouse model for Duchenne muscular dystrophy. Regen Med. 2007;2(3):275–288.

74. OptiStem.org [homepage on the Internet]. Progress reports. OtpiStem; 2012 [cited August 8, 2012]. Available from: http://www.optistem.org/progress-reports. Accessed August 18, 2012.

75. Harper SQ, Hauser MA, DelloRusso C, et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med. 2002;8(3):253–261.

76. Sakamoto M, Yuasa K, Yoshimura M, et al. Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene. Biochem Biophys Res Commun. 2002;293(4): 1265–1272.

77. Bowles DE, McPhee SW, Li C, et al. Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther. 2012;20(2):443–455.

78. Seto JT, Ramos JN, Muir L, Chamberlain JS, Odom GL. Gene replace-ment therapies for duchenne muscular dystrophy using adeno-associated viral vectors. Curr Gene Ther. 2012;12(3):139–151.

79. Yuasa K, Yoshimura M, Urasawa N, et al. Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther. 2007;14(17):1249–1260.

80. Koo T, Malerba A, Athanasopoulos T, et al. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of alpha1-syntrophin and alpha-dystrobrevin in skeletal muscles of mdx mice. Hum Gene Ther. 2011;22(11): 1379–1388.

81. Kornegay JN, Li J, Bogan JR, et al. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neo-natal dystrophin-deficient dogs. Mol Ther. 2010;18(8):1501–1508.

82. Ohshima S, Shin JH, Yuasa K, et al. Transduction efficiency and immune response associated with the administration of AAV8 vector into dog skeletal muscle. Mol Ther. 2009;17(1):73–80.

83. Mendell JR, Campbell K, Rodino-Klapac L, et al. Dystrophin immu-nity in Duchenne’s muscular dystrophy. N Engl J Med. 2010;363(15): 1429–1437.

84. Acsadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature. 1991;352(6338):815–818.

85. Danko I, Fritz JD, Latendresse JS, Herweijer H, Schultz E, Wolff JA. Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection. Hum Mol Genet. 1993;2(12):2055–2061.

86. Decrouy A, Renaud JM, Davis HL, Lunde JA, Dickson G, Jasmin BJ. Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability. Gene Ther. 1997;4(5): 401–408.

87. Pichavant C, Chapdelaine P, Cerri DG, Bizario JC, Tremblay JP. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles. Hum Gene Ther. 2010;21(11):1591–1601.

88. Romero NB, Braun S, Benveniste O, et al. Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy. Hum Gene Ther. 2004;15(11):1065–1076.

89. Duan D. Myodys, a full-length dystrophin plasmid vector for Duchenne and Becker muscular dystrophy gene therapy. Curr Opin Mol Ther. 2008;10(1):86–94.

90. Bachrach E, Li S, Perez AL, et al. Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle pro-genitor cells. Proc Natl Acad Sci U S A. 2004;101(10):3581–3586.

91. Pichavant C, Chapdelaine P, Cerri DG, et al. Expression of dog microdystrophin in mouse and dog muscles by gene therapy. Mol Ther. 2010;18(5):1002–1009.

92. Kimura E, Li S, Gregorevic P, Fall BM, Chamberlain JS. Dystrophin delivery to muscles of mdx mice using lentiviral vectors leads to myogenic progenitor targeting and stable gene expression. Mol Ther. 2010;18(1):206–213.

93. Sampaolesi M, Blot S, D’Antona G, et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature. 2006; 444(7119):574–579.

94. Feng SW, Chen F, Cao J, et al. Restoration of muscle fibers and satellite cells after isogenic MSC transplantation with microdystrophin gene delivery. Biochem Biophys Res Commun. 2012;419(1):1–6.

95. Ikezawa M, Cao B, Qu Z, et al. Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation. Hum Gene Ther. 2003;14(16):1535–1546.

96. Moisset PA, Skuk D, Asselin I, et al. Successful transplantation of genetically corrected DMD myoblasts following ex vivo transduction with the dystrophin minigene. Biochem Biophys Res Commun. 1998; 247(1):94–99.

97. Moisset PA, Gagnon Y, Karpati G, Tremblay JP. Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts. Gene Ther. 1998;5(10):1340–1346.

98. Hoshiya H, Kazuki Y, Abe S, et al. A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene. Mol Ther. 2009;17(2):309–317.

99. Nakahata T, Awaya T, Chang H, et al. Derivation of engraftable myogenic precursors from murine ES/iPS cells and generation of disease-specific iPS cells from patients with Duchenne muscular dystrophy (DMD) and other diseases. Rinsho Shinkeigaku. 2010; 50(11):889.

100. Kazuki Y, Hiratsuka M, Takiguchi M, et al. Complete genetic correction of ips cells from Duchenne muscular dystrophy. Mol Ther. 2010;18(2):386–393.

101. Tedesco FS, Hoshiya H, D’Antona G, et al. Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy. Sci Transl Med. 2011;3(96):96ra78.


Dovepress

Dovepress

159


http://clinicaltrials.gov/ct2/results?term=dmd

http://clinicaltrials.gov/ct2/results?term=dmd

http://www.optistem.org/progress-reports

http://www.optistem.org/progress-reports

www.dovepress.com

www.dovepress.com

www.dovepress.com


102. Rando TA, Disatnik MH, Zhou LZ. Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc Natl Acad Sci U S A. 2000;97(10):5363–5368.

103. Bertoni C, Rando TA. Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides. Hum Gene Ther. 2002;13(6):707–718.

104. Bartlett RJ, Stockinger S, Denis MM, et al. In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotechnol. 2000;18(6):615–622.

105. Alexeev V, Igoucheva O, Yoon K. Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides. Gene Ther. 2002;9(24):1667–1675.

106. Bertoni C, Lau C, Rando TA. Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping. Hum Mol Genet. 2003;12(10):1087–1099.

107. Bertoni C, Morris GE, Rando TA. Strand bias in oligonucleotide- mediated dystrophin gene editing. Hum Mol Genet. 2005;14(2):221–233.

108. Maguire K, Suzuki T, DiMatteo D, Parekh-Olmedo H, Kmiec E. Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice. BMC Mol Biol. 2009; 10:15.

109. Kole R, Sazani P. Antisense effects in the cell nucleus: modification of splicing. Curr Opin Mol Ther. 2001;3(3):229–234.

110. Benchaouir R, Goyenvalle A. Splicing modulation mediated by small nuclear RNAs as therapeutic approaches for muscular dystrophies. Curr Gene Ther. 2012;12(3):179–191.

111. Dunckley MG, Manoharan M, Villiet P, Eperon IC, Dickson G. Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet. 1998;7(7): 1083–1090.

112. Mann CJ, Honeyman K, Cheng AJ, et al. Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci U S A. 2001;98(1):42–47.

113. Arechavala-Gomeza V, Anthony K, Morgan J, Muntoni F. Antisense oligonucleotide-mediated exon skipping for duchenne muscular dystrophy: progress and challenges. Curr Gene Ther. 2012;12(3): 152–160.

114. Lu QL, Rabinowitz A, Chen YC, et al. Systemic delivery of anti-sense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci U S A. 2005;102(1): 198–203.

115. Aoki Y, Nakamura A, Yokota T, et al. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther. 2010;18(11):1995–2005.

116. Yokota T, Hoffman E, Takeda S. Antisense oligo-mediated multiple exon skipping in a dog model of duchenne muscular dystrophy. Methods Mol Biol. 2011;709:299–312.

117. Mitrpant C, Fletcher S, Iversen PL, Wilton SD. By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping. J Gene Med. 2009;11(1):46–56.

118. Yin H, Lu Q, Wood M. Effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice. Mol Ther. 2008;16(1):38–45.

119. Prosensa.eu [homepage on the Internet]. Pre-clinical portfolio. Leiden, The Netherlands: Prosensa; 2012 [cited August 8, 2012]. Available from: http://prosensa.eu/technology-and-products/pre-clinical-portfolio. Accessed August 18, 2012.

120. van Deutekom JC, Janson AA, Ginjaar IB, et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med. 2007;357(26):2677–2686.

121. Neri M, Torelli S, Brown S, et al. Dystrophin levels as low as 30% are sufficient to avoid muscular dystrophy in the human. Neuromuscul Disord. 2007;17(11–12):913–918.

122. Goemans NM, Tulinius M, van den Akker JT, et al. Systemic administration of PRO051 in Duchenne’s muscular dystrophy. N Engl J Med. 2011;364(16):1513–1522.

123. Sareptatherapeutics.com [homepage on the Internet]. Duchenne muscular dystrophy: drug candidates/clinical trials. Cambridge, MA: Serepta Therapeutics; 2012 [cited August 8, 2012]. Available from: http://www.sareptatherapeutics.com/our-programs/rare-diseases/duchenne-muscular-dystrophy. Accessed August 18, 2012.

124. Arechavala-Gomeza V, Graham IR, Popplewell LJ, et al. Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle. Hum Gene Ther. 2007;18(9):798–810.

125. Kinali M, Arechavala-Gomeza V, Feng L, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 2009;8(10): 918–928.

126. Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011;378(9791):595–605.

127. De Angelis FG, Sthandier O, Berarducci B, et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48–50 DMD cells. Proc Natl Acad Sci U S A. 2002;99(14):9456–9461.

128. Brun C, Suter D, Pauli C, et al. U7snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping. Cell Mol Life Sci. 2003;60(3):557–566.

129. Goyenvalle A, Vulin A, Fougerousse F, et al. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science. 2004; 306(5702):1796–1799.

130. Denti MA, Rosa A, D’Antona G, et al. Chimeric adeno-associated virus/antisense U1 small nuclear RNA effectively rescues dystrophin synthesis and muscle function by local treatment of mdx mice. Hum Gene Ther. 2006;17(5):565–574.

131. Goyenvalle A, Babbs A, Wright J, et al. Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping. Hum Mol Genet. Mar 13, 2012;21(11): 2559–2571.

132. Goyenvalle A, Wright J, Babbs A, Wilkins V, Garcia L, Davies KE. Engineering multiple U7snRNA constructs to induce single and multiexon-skipping for Duchenne muscular dystrophy. Mol Ther. 2012;20(6):1212–1221.

133. Bish LT, Sleeper MM, Forbes SC, et al. Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping. Mol Ther. 2012;20(3): 580–589.

134. Barbash IM, Cecchini S, Faranesh AZ, et al. MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Gene Ther. Epub May 3, 2012.

135. Chapdelaine P, Pichavant C, Rousseau J, Paques F, Tremblay JP. Meganucleases can restore the reading frame of a mutated dystrophin. Gene Ther. 2010;17(7):846–858.

136. Aartsma-Rus A, Fokkema I, Verschuuren J, et al. Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat. 2009;30(3):293–299.

137. Heemskerk H, de Winter C, van Kuik P, et al. Preclinical PK and PD studies on 2′-O-methyl-phosphorothioate RNA antisense oligonucleotides in the mdx mouse model. Mol Ther. 2010;18(6): 1210–1217.

138. Aartsma-Rus A, van Ommen GJ. Less is more: therapeutic exon skipping for Duchenne muscular dystrophy. Lancet Neurol. 2009; 8(10):873–875.

139. ‘t Hoen PA, de Meijer EJ, Boer JM, et al. Generation and characterization of transgenic mice with the full-length human DMD gene. J Biol Chem. 2008;283(9):5899–5907.


Dovepress

Dovepress

160

Perkins and Davies

http://prosensa.eu/technology-and-products/pre-clinical-portfolio

http://prosensa.eu/technology-and-products/pre-clinical-portfolio

http://www.sareptatherapeutics.com/our-programs/rare-diseases/duchenne-muscular-dystrophy

http://www.sareptatherapeutics.com/our-programs/rare-diseases/duchenne-muscular-dystrophy

www.dovepress.com

www.dovepress.com

www.dovepress.com


140. Mitrpant C, Adams AM, Meloni PL, Muntoni F, Fletcher S, Wilton SD. Rational design of antisense oligomers to induce dystrophin exon skipping. Mol Ther. 2009;17(8):1418–1426.

141. O’Leary DA, Sharif O, Anderson P, et al. Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening. PLoS One. 2009;4(12): e8348.

142. Moulton HM. Cell-penetrating peptides enhance systemic delivery of antisense morpholino oligomers. Methods Mol Biol. 2012;867: 407–414.

143. Malik V, Rodino-Klapac LR, Viollet L, et al. Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy. Ann Neurol. 2010;67(6):771–780.

144. Bushby K. Genetics and the muscular dystrophies. Dev Med Child Neurol. 2000;42(11):780–784.

145. Manuvakhova M, Keeling K, Bedwell DM. Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA. 2000;6(7):1044–1055.

146. Palmer E, Wilhelm JM, Sherman F. Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics. Nature. 1979;277(5692):148–150.

147. Barton-Davis ER, Cordier L, Shoturma DI, Leland SE, Sweeney HL. Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J Clin Invest. 1999;104(4):375–381.

148. Loufrani L, Dubroca C, You D, et al. Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin. Arterioscler Thromb Vasc Biol. 2004;24(4):671–676.

149. Politano L, Nigro G, Nigro V, et al. Gentamicin administration in Duchenne patients with premature stop codon. Preliminary results. Acta Myol. 2003;22(1):15–21.

150. Wagner KR, Hamed S, Hadley DW, et al. Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations. Ann Neurol. 2001;49(6):706–711.

151. Yoshizawa S, Fourmy D, Puglisi JD. Structural origins of gentamicin antibiotic action. EMBO J. 1998;17(22):6437–6448.

152. Dunant P, Walter MC, Karpati G, Lochmuller H. Gentamicin fails to increase dystrophin expression in dystrophin-deficient muscle. Muscle Nerve. 2003;27(5):624–627.

153. Yukihara M, Ito K, Tanoue O, et al. Effective drug delivery system for duchenne muscular dystrophy using hybrid liposomes including gentamicin along with reduced toxicity. Biol Pharm Bull. 2011;34(5): 712–716.

154. A New Approach to Drug Discovery [webpage on the Internet]. Therapeutic areas. South Plainfield, NJ: PTC Therapeutics; 2012 [cited August 8, 2012]. Available from: http://www.ptcbio.com/therapeutic_areas. Accessed August 18, 2012.

155. Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature. 2007;447(7140):87–91.

156. Arakawa M, Shiozuka M, Nakayama Y, et al. Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice. J Biochem. 2003;134(5):751–758.

157. Love DR, Hill DF, Dickson G, et al. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature. 1989;339(6219): 55–58.

158. Tinsley JM, Blake DJ, Roche A, et al. Primary structure of dystrophin-related protein. Nature. 1992;360(6404):591–593.

159. Khurana TS, Watkins SC, Chafey P, et al. Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromuscul Disord. 1991;1(3):185–194.

160. Fisher R, Tinsley JM, Phelps SR, et al. Non-toxic ubiquitous over-expression of utrophin in the mdx mouse. Neuromuscul Disord. 2001;11(8):713–721.

161. Moorwood C, Lozynska O, Suri N, Napper AD, Diamond SL, Khurana TS. Drug discovery for Duchenne muscular dystrophy via utrophin promoter activation screening. PLoS One. 2011;6(10):e26169.

162. Fairclough RJ, Perkins KJ, Davies KE. Pharmacologically targeting the primary defect and downstream pathology in duchenne muscular dystrophy. Curr Gene Ther. 2012;12(3):206–244.

163. Gramolini AO, Belanger G, Thompson JM, Chakkalakal JV, Jasmin BJ. Increased expression of utrophin in a slow vs a fast muscle involves posttranscriptional events. Am J Physiol Cell Physiol. 2001;281(4): C1300–C1309.

164. Selsby JT, Morine KJ, Pendrak K, Barton ER, Sweeney HL. Rescue of dystrophic skeletal muscle by PGC-1alpha involves a fast to slow fiber type shift in the mdx mouse. PLoS One. 2012;7(1):e30063.

165. Handschin C, Kobayashi YM, Chin S, Seale P, Campbell KP, Spiegelman BM. PGC-1alpha regulates the neuromuscular junction program and ameliorates Duchenne muscular dystrophy. Genes Dev. 2007;21(7):770–783.

166. Angus LM, Chakkalakal JV, Mejat A, et al. Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1{alpha}, drives utrophin gene expression at the neuromuscular junction. Am J Physiol Cell Physiol. 2005;289(4):C908–C917.

167. Miura P, Chakkalakal JV, Boudreault L, et al. Pharmacological activation of PPARbeta/delta stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice. Hum Mol Genet. 2009;18(23):4640–4649.

168. Lampen A, Siehler S, Ellerbeck U, Gottlicher M, Nau H. New molecular bioassays for the estimation of the teratogenic potency of valproic acid derivatives in vitro: activation of the peroxisomal proliferator-activated receptor (PPARdelta). Toxicol Appl Pharmacol. 1999;160(3):238–249.

169. Lopez-Soriano J, Chiellini C, Maffei M, Grimaldi PA, Argiles JM. Roles of skeletal muscle and peroxisome proliferator-activated receptors in the development and treatment of obesity. Endocr Rev. 2006;27(3):318–329.

170. Gardner OS, Dewar BJ, Graves LM. Activation of mitogen-activated protein kinases by peroxisome proliferator-activated receptor ligands: an example of nongenomic signaling. Mol Pharmacol. 2005;68(4): 933–941.

171. Balakumar P, Kathuria S. Submaximal PPARgamma activation and endothelial dysfunction: new perspectives for the management of cardiovascular disorders. Br J Pharmacol. 2012;166(7):1981–1992.

172. Leick L, Fentz J, Bienso RS, et al. PGC-1{alpha} is required for AICAR-induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2010;299(3): E456–E465.

173. Ljubicic V, Khogali S, Renaud JM, Jasmin BJ. Chronic AMPK stimulation attenuates adaptive signaling in dystrophic skeletal muscle. Am J Physiol Cell Physiol. 2011;302(1):C110–C121.

174. Pold R, Jensen LS, Jessen N, et al. Long-term AICAR administration and exercise prevents diabetes in ZDF rats. Diabetes. 2005;54(4): 928–934.

175. Passananti C, Corbi N, Onori A, Di Certo MG, Mattei E. Transgenic mice expressing an artificial zinc finger regulator targeting an endogenous gene. Methods Mol Biol. 2010;649:183–206.

176. Tinsley JM, Fairclough RJ, Storer R, et al. Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse. PLoS One. 2011; 6(5):e19189.

177. Biomarin Pharmaceutical Inc. BioMarin and Summit plc sign worldwide licensing agreement for Duchenne muscular dystrophy program [press release]. Novato, CA: Biomarin Pharmaceutical Inc; 2008 [Jul 22]. Available from: http://phx.corporate-ir.net/phoenix.zhtml?c=106657&p=irol-newsArticle&ID=1177621&highlight=. Accessed July 22, 2008.

178. Fairclough RF, Squire SE, Potter AC, et al. Identification of new chemical compounds which upregulate utrophin for the therapy of Duchenne muscular dystrophy. Proceedings of the Muscular Dystrophy Campaign UK Neuromuscular Translational Research Conference; 2012 Mar 22–23; Newcastle, UK.


Dovepress

Dovepress

161


http://www.ptcbio.com/therapeutic_areas

http://www.ptcbio.com/therapeutic_areas

http://phx.corporate-ir.net/phoenix.zhtml?c=106657&p=irol-newsArticle&ID=1177621&highlight=


www.dovepress.com

www.dovepress.com

www.dovepress.com


179. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999;285(5433):1569–1572.

180. Sonnemann KJ, Heun-Johnson H, Turner AJ, Baltgalvis KA, Lowe DA, Ervasti JM. Functional substitution by TAT-utrophin in dystrophin-deficient mice. PLoS Med. 2009;6(5):e1000083.

181. Call JA, Ervasti JM, Lowe DA. TAT-muUtrophin mitigates the pathophysiology of dystrophin and utrophin double-knockout mice. J Appl Physiol. 2011;111(1):200–205.

182. Retrophin. RE-001: Retrophin’s investigational agent for Duchenne muscular dystrophy. New York, NY: Retrophin LLC; 2011 [cited August 8, 2012]. Available from: http://www.retrophin.com/pipeline.php. Accessed September 25, 2012.

183. Tivorsan Pharmaceuticals. MDA awards $1 million to Tivorsan Pharmaceuticals for accelerating pivotal pre-clinical work on Tvn-102 as a potential muscular dystrophy treatment [press release]. Providence, RI: Tivorsan Pharmaceuticals, Inc; 2012 [Jan 5]. Available from: http://www.tivorsan.com/wp-content/uploads/2012/01/PR_MDATivorsan_1M_Final_1_5_12.pdf. Accessed January 5, 2012.

184. Zanotti S, Negri T, Cappelletti C, et al. Decorin and biglycan expression is differentially altered in several muscular dystrophies. Brain. 2005;128(Pt 11):2546–2555.

185. Amenta AR, Yilmaz A, Bogdanovich S, et al. Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice. Proc Natl Acad Sci U S A. 2010;108(2):762–767.

186. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69(1):11–25.

187. Hodges BL, Hayashi YK, Nonaka I, Wang W, Arahata K, Kaufman SJ. Altered expression of the alpha7beta1 integrin in human and murine muscular dystrophies. J Cell Sci. 1997;110(Pt 22):2873–2881.

188. Mayer U, Saher G, Fassler R, et al. Absence of integrin alpha 7 causes a novel form of muscular dystrophy. Nat Genet. 1997;17(3):318–323.

189. Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ. Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol. 2001;152(6):1207–1218.

190. Liu J, Burkin DJ, Kaufman SJ. Increasing alpha 7 beta 1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression. Am J Physiol Cell Physiol. 2008;294(2):C627–C640.

191. Gurpur PB, Liu J, Burkin DJ, Kaufman SJ. Valproic acid activates the PI3K/Akt/mTOR pathway in muscle and ameliorates pathology in a mouse model of Duchenne muscular dystrophy. Am J Pathol. 2009;174(3):999–1008.

192. Bodine SC, Stitt TN, Gonzalez M, et al. Akt/mTOR pathway is a cru-cial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3(11):1014–1019.

193. Rooney JE, Gurpur PB, Burkin DJ. Laminin-111 protein therapy pre-vents muscle disease in the mdx mouse model for Duchenne muscular dystrophy. Proc Natl Acad Sci U S A. 2009;106(19):7991–7996.

194. Rooney JE, Gurpur PB, Yablonka-Reuveni Z, Burkin DJ. Laminin-111 restores regenerative capacity in a mouse model for alpha7 integrin congenital myopathy. Am J Pathol. 2009;174(1):256–264.

195. Coral-Vazquez R, Cohn RD, Moore SA, et al. Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy. Cell. 1999;98(4):465–474.

196. Prothelia [webpage on the Internet]. Therapeutics for muscular dystrophy. Milford, MA: Prothelia Inc.; 2012 [cited August 8, 2012]. Available from: http://www.prothelia.com/livesite/pages/pipeline. Accessed September 25, 2012.

197. CombinatoRx. CombinatoRx and Charley’s Fund/Nash Avery Foun-dation collaborate to develop novel agents for Duchenne muscular dystrophy [press release]. CombinatoRx, Incorporated; 2007 [cited August 8, 2012]. Available from: http://phx.corporate-ir.net/phoenix.zhtml?c=148036&p=irol-newsArticle&ID=1074719&highlight=. Accessed September 25, 2012.

198. Rando TA. Oxidative stress and the pathogenesis of muscular dystrophies. Am J Phys Med Rehabil. 2002;81(Suppl 11):S175–S186.

199. Morgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res. 2011;21(1):103–115.

200. Spurney CF, Guerron AD, Yu Q, et al. Membrane sealant Poloxamer P188 protects against isoproterenol induced cardiomyopathy in dystrophin deficient mice. BMC Cardiovasc Disord. 2011;11:20.

201. Townsend D, Turner I, Yasuda S, et al. Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs. J Clin Invest. 2010;120(4):1140–1150.

202. Tidball JG, Wehling-Henricks M. The role of free radicals in the pathophysiology of muscular dystrophy. J Appl Physiol. 2007;102(4): 1677–1686.

203. Hankard R, Mauras N, Hammond D, Haymond M, Darmaun D. Is glutamine a ‘conditionally essential’ amino acid in Duchenne muscular dystrophy? Clin Nutr. 1999;18(6):365–369.

204. Salehian B, Mahabadi V, Bilas J, Taylor WE, Ma K. The effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression. Metabolism. 2006; 55(9):1239–1247.

205. Mok E, Constantin B, Favreau F, et al. l-Glutamine administration reduces oxidized glutathione and MAP kinase signaling in dystrophic muscle of mdx mice. Pediatr Res. 2008;63(3):268–273.

206. Hibaoui Y, Reutenauer-Patte J, Patthey-Vuadens O, Ruegg UT, Dor-chies OM. Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy. J Pineal Res. 2011;51(2):163–171.

207. Chahbouni M, Escames G, Venegas C, et al. Melatonin treatment normalizes plasma pro-inflammatory cytokines and nitrosative/ oxidative stress in patients suffering from Duchenne muscular dystrophy. J Pineal Res. 2010;48(3):282–289.

208. Sah JF, Balasubramanian S, Eckert RL, Rorke EA. Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases. J Biol Chem. 2004;279(13):12755–12762.

209. Chen PC, Wheeler DS, Malhotra V, Odoms K, Denenberg AG, Wong HR. A green tea-derived polyphenol, epigallocatechin-3-gallate, inhibits IkappaB kinase activation and IL-8 gene expression in respira-tory epithelium. Inflammation. 2002;26(5):233–241.

210. Buyse GM, Goemans N, van den Hauwe M, et al. Idebenone as a novel, therapeutic approach for Duchenne muscular dystrophy: results from a 12 month, double-blind, randomized placebo-controlled trial. Neuromuscul Disord. 2011;21(6):396–405.

211. Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med. 1989;6(6):593–597.

212. Whitehead NP, Pham C, Gervasio OL, Allen DG. N-Acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice. J Physiol. 2008;586(7):2003–2014.

213. Ter rill JR, Radley-Crabb HG, Grounds MD, Arthur PG. N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis. Neuromuscul Disord. 2011;22(5):427–434.

214. Smith RA, Murphy MP. Mitochondria-targeted antioxidants as therapies. Discov Med. 2011;11(57):106–114.

215. Gehrig SM, van der Poel C, Sayer TA, et al. Hsp72 preserves muscle function and slows progression of severe muscular dystrophy. Nature. 2012;484(7394):394–398.

216. Lemasters JJ, Theruvath TP, Zhong Z, Nieminen AL. Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta. 2009;1787(11):1395–1401.

217. McGuinness O, Yafei N, Costi A, Crompton M. The presence of two classes of high-affinity cyclosporin A binding sites in mitochondria. Evidence that the minor component is involved in the opening of an inner-membrane Ca(2+)-dependent pore. Eur J Biochem. 1990;194(2): 671–679.


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218. Matas J, Young NT, Bourcier-Lucas C, et al. Increased expression and intramitochondrial translocation of cyclophilin-D associates with increased vulnerability of the permeability transition pore to stress-induced opening during compensated ventricular hypertrophy. J Mol Cell Cardiol. 2009;46(3):420–430.

219. De Luca A, Nico B, Liantonio A, et al. A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice. Am J Pathol. 2005;166(2):477–489.

220. Weller B, Massa R, Karpati G, Carpenter S. Glucocorticoids and immunosuppressants do not change the prevalence of necrosis and regeneration in mdx skeletal muscles. Muscle Nerve. 1991;14(8): 771–774.

221. Sharma KR, Mynhier MA, Miller RG. Cyclosporine increases muscular force generation in Duchenne muscular dystrophy. Neurology. 1993;43(3 Pt 1):527–532.

222. Kirschner J, Schessl J, Schara U, et al. Treatment of Duchenne muscular dystrophy with ciclosporin A: a randomised, double-blind, placebo-controlled multicentre trial. Lancet Neurol. 2010;9(11): 1053–1059.

223. Khairallah M, Khairallah RJ, Young ME, et al. Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency. Proc Natl Acad Sci U S A. 2008;105(19):7028–7033.

224. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev. 2001;81(1):209–237.

225. Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol. 2001;155(1):123–131.

226. Brunelli S, Sciorati C, D’Antona G, et al. Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy. Proc Natl Acad Sci U S A. 2007;104(1):264–269.

227. Wang G, Burczynski FJ, Hasinoff BB, Zhang K, Lu Q, Anderson JE. Development of a nitric oxide-releasing analogue of the muscle relaxant guaifenesin for skeletal muscle satellite cell myogenesis. Mol Pharm. 2009;6(3):895–904.

228. Sciorati C, Galvez BG, Brunelli S, et al. Ex vivo treatment with nitric oxide increases mesoangioblast therapeutic efficacy in muscular dystrophy. J Cell Sci. 2006;119(Pt 24):5114–5123.

229. Takimoto E, Champion HC, Li M, et al. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med. 2005;11(2):214–222.

230. Asai A, Sahani N, Kaneki M, Ouchi Y, Martyn JA, Yasuhara SE. Primary role of functional ischemia, quantitative evidence for the two-hit mechanism, and phosphodiesterase-5 inhibitor therapy in mouse muscular dystrophy. PLoS One. 2007;2(8):e806.

231. Adamo CM, Dai DF, Percival JM, et al. Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A. 2010;107(44):19079–19083.

232. Mok E, Letellier G, Cuisset JM, et al. Lack of functional benefit with glutamine versus placebo in Duchenne muscular dystrophy: a randomized crossover trial. PLoS One. 2009;4(5):e5448.

233. Kumamoto T, Fujimoto S, Ito T, Horinouchi H, Ueyama H, Tsuda T. Proteasome expression in the skeletal muscles of patients with muscular dystrophy. Acta Neuropathol. 2000;100(6):595–602.

234. Tidball JG, Spencer MJ. Calpains and muscular dystrophies. Int J Biochem Cell Biol. 2000;32(1):1–5.

235. Sawada H, Nagahiro K, Kikukawa Y, et al. Therapeutic effect of camostat mesilate on Duchenne muscular dystrophy in mdx mice. Biol Pharm Bull. 2003;26(7):1025–1027.

236. Briguet A, Erb M, Courdier-Fruh I, et al. Effect of calpain and proteasome inhibition on Ca2+-dependent proteolysis and muscle histopathology in the mdx mouse. FASEB J. Dec 2008;22(12):4190–4200.

237. Childers MK, Bogan JR, Bogan DJ, et al. Chronic administration of a leupeptin-derived calpain inhibitor fails to ameliorate severe muscle pathology in a canine model of duchenne muscular dystrophy. Front Pharmacol. 2011;2:89.

238. Bonuccelli G, Sotgia F, Schubert W, et al. Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. Am J Pathol. 2003;163(4):1663–1675.

239. Bonuccelli G, Sotgia F, Capozza F, Gazzerro E, Minetti C, Lisanti MP. Localized treatment with a novel FDA-approved proteasome inhibitor blocks the degradation of dystrophin and dystrophin-associated proteins in mdx mice. Cell Cycle. 2007;6(10):1242–1248.

240. Gazzerro E, Assereto S, Bonetto A, et al. Therapeutic potential of proteasome inhibition in Duchenne and Becker muscular dystrophies. Am J Pathol. 2010;176(4):1863–1877.

241. Assereto S, Stringara S, Sotgia F, et al. Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment. Am J Physiol Cell Physiol. 2006;290(2):C577–C582.

242. Acharyya S, Villalta SA, Bakkar N, et al. Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J Clin Invest. 2007;117(4):889–901.

243. Porreca E, Guglielmi MD, Uncini A, et al. Haemostatic abnormalities, cardiac involvement and serum tumor necrosis factor levels in X-linked dystrophic patients. Thromb Haemost. 1999;81(4):543–546.

244. Whitehead NP, Yeung EW, Allen DG. Muscle damage in mdx (dystrophic) mice: role of calcium and reactive oxygen species. Clin Exp Pharmacol Physiol. 2006;33(7):657–662.

245. De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, Fiers W, Haegeman G. Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation. Proc Natl Acad Sci U S A. 1997;94(25): 13504–13509.

246. Pan Y, Chen C, Shen Y, et al. Curcumin alleviates dystrophic muscle pathology in mdx mice. Mol Cells. 2008;25(4):531–537.

247. Durham WJ, Arbogast S, Gerken E, Li YP, Reid MB. Progressive nuclear factor-kappaB activation resistant to inhibition by contraction and curcumin in mdx mice. Muscle Nerve. 2006;34(3): 298–303.

248. Tang Y, Reay DP, Salay MN, et al. Inhibition of the IKK/NF-kappaB pathway by AAV gene transfer improves muscle regeneration in older mdx mice. Gene Ther. 2010;17(12):1476–1483.

249. Peterson JM, Kline W, Canan BD, et al. Peptide-based inhibition of NF-kappaB rescues diaphragm muscle contractile dysfunction in a murine model of Duchenne muscular dystrophy. Mol Med. 2011;17(5–6):508–515.

250. Tilstra J, Rehman KK, Hennon T, Plevy SE, Clemens P, Robbins PD. Protein transduction: identification, characterization and optimization. Biochem Soc Trans. 2007;35(Pt 4):811–815.

251. Reay DP, Yang M, Watchko JF, et al. Systemic delivery of NEMO binding domain/IKKgamma inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology. Neurobiol Dis. 2011;43(3):598–608.

252. Reay DP, Niizawa GA, Watchko JF, et al. Effect of NF-kappaB inhibition on AAV9 minidystrophin gene transfer to the mdx mouse. Mol Med. 2012;18(1):466–476.

253. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004;18(7):816–827.

254. Bernasconi P, Torchiana E, Confalonieri P, et al. Expression of transforming growth factor-beta 1 in dystrophic patient muscles correlates with fibrosis. Pathogenetic role of a fibrogenic cytokine. J Clin Invest. 1995;96(2):1137–1144.

255. Burks TN, Cohn RD. Role of TGF-beta signaling in inherited and acquired myopathies. Skelet Muscle. 2011;1(1):19.

256. Bizario JC, Cerri DG, Rodrigues LC, et al. Imatinib mesylate ameliorates the dystrophic phenotype in exercised mdx mice. J Neuroimmunol. 2009;212(1–2):93–101.

257. Huang P, Zhao XS, Fields M, Ransohoff RM, Zhou L. Imatinib attenuates skeletal muscle dystrophy in mdx mice. FASEB J. 2009; 23(8):2539–2548.


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258. Morris CA, Selsby JT, Morris LD, Pendrak K, Sweeney HL. Bowman-Birk inhibitor attenuates dystrophic pathology in mdx mice. J Appl Physiol. 2010;109(5):1492–1499.

259. Roffe S, Hagai Y, Pines M, Halevy O. Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: effect on myotube fusion. Exp Cell Res. 2010;316(6): 1061–1069.

260. Turgeman T, Hagai Y, Huebner K, et al. Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone. Neuromuscul Disord. 2008;18(11):857–868.

261. Huebner KD, Jassal DS, Halevy O, Pines M, Anderson JE. Functional resolution of fibrosis in mdx mouse dystrophic heart and skeletal muscle by halofuginone. Am J Physiol Heart Circ Physiol. 2008;294(4):H1550–H1561.

262. Gosselin LE, Williams JE, Deering M, Brazeau D, Koury S, Martinez DA. Localization and early time course of TGF-beta 1 mRNA expression in dystrophic muscle. Muscle Nerve. 2004;30(5):645–653.

263. Shi S, Hoogaars WM, de Gorter DJ, et al. BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model. Neurobiol Dis. 2011;41(2):353–360.

264. Cohn RD, van Erp C, Habashi JP, et al. Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states. Nat Med. 2007;13(2):204–210.

265. Spurney CF, Sali A, Guerron AD, et al. Losartan decreases cardiac muscle fibrosis and improves cardiac function in dystrophin-deficient mdx mice. J Cardiovasc Pharmacol Ther. 2011;16(1):87–95.

266. Nelson CA, Hunter RB, Quigley LA, et al. Inhibiting TGF-beta activity improves respiratory function in mdx mice. Am J Pathol. 2011;178(6): 2611–2621.

267. Rafael-Fortney JA, Chimanji NS, Schill KE, et al. Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice. Circulation. 2011;124(5): 582–588.

268. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997;387(6628):83–90.

269. Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem. 2002;277(51):49831–49840.

270. Wahl M. ACE-031 clinical trials in Duchenne MD stopped for now. Tucson, AZ: Quest; 2011 [cited August 8, 2012]. Available from: http://quest.mda.org/news/ace-031-clinical-trials-duchenne-md-stopped-now. Accessed August 18, 2012.

271. Wagner KR, Fleckenstein JL, Amato AA, et al. A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy. Ann Neurol. 2008;63(5):561–571.

272. Qiao C, Li J, Zheng H, et al. Hydrodynamic limb vein injection of adeno-associated virus serotype 8 vector carrying canine myostatin propeptide gene into normal dogs enhances muscle growth. Hum Gene Ther. 2009;20(1):1–10.

273. Kang JK, Malerba A, Popplewell L, Foster K, Dickson G. Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa-guanidine morpholino oligomer treatment. Mol Ther. 2011;19(1):159–164.

274. Kemaladewi DU, Hoogaars WM, van Heiningen SH, et al. Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystro-phy. BMC Med Genomics. 2011;4:36.

275. Barton-Davis ER, Shoturma DI, Musaro A, Rosenthal N, Sweeney HL. Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci U S A. 1998;95(26):15603–15607.

276. Gregorevic P, Plant DR, Leeding KS, Bach LA, Lynch GS. Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration. Am J Pathol. 2002;161(6):2263–2272.

277. Gehrig SM, van der Poel C, Hoeflich A, Naim T, Lynch GS, Metzger F. Therapeutic potential of PEGylated insulin-like growth factor I for skeletal muscle disease evaluated in two murine models of muscular dystrophy. Growth Horm IGF Res. 2012;22(2):69–75.

278. Abmayr S, Gregorevic P, Allen JM, Chamberlain JS. Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery. Mol Ther. 2005;12(3):441–450.


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